1. Gusobanukirwa kwambere

Kuri iki cyiciro, dukeneye gusobanukirwa bimwe na terminologiya, kugirango twirinde gukora amakosa imbere ya bakuru bacu, nka:

Ikibazo: Ni irihe tandukaniro riri hagati ya RT-PCR, qPCR, Igihe nyacyo PCR, nigihe nyacyo RT-PCR?

Igisubizo: RT-PCR isubiza inyuma PCR.Muri RT-PCR, umugozi wa RNA uhindurwa muri ADN yuzuzanya, hanyuma igakoreshwa nk'icyitegererezo cyo kongera ADN na PCR.
Igihe nyacyo-PCR na qPCR.

Nubwo byombi PCR-nyayo (igihe nyacyo-fluorescent quantitative PCR) hamwe na Reverse transcription PCR (reverse transcript PCR) bisa nkaho ari impfunyapfunyo nka RT-PCR, amasezerano mpuzamahanga ni: RT-PCR yerekeza cyane cyane kuri transcriptPCR, Igihe nyacyo PCR mu magambo ahinnye muri rusange ni qPCR (umubare wuzuye-igihe PCR).

Kandi igihe-nyacyo RT-PCR (RT-qPCR), Nibisubiranamo PCR ihujwe na tekinoroji ya fluorescent: banza ubone cDNA (RT) muri RNA ihinduranya inyandiko, hanyuma ukoreshe PCR-nyayo kugirango isesengure ryinshi (qPCR).Laboratoire nyinshi zikora RT-qPCR, ni ukuvuga, ubushakashatsi ku mvugo ya RNA kumanura-amabwiriza, bityo qPCR abantu bose bavuga muri laboratoire yerekeza kuri RT-qPCR, ariko ntiwibagirwe ko hakiri ibizamini byinshi bya ADN mubisabwa kwa muganga.Isesengura ryinshi, nka virusi ya hepatite B kumenya HBV.

Ikibazo: Nyuma yo gusoma ibintu byinshi bya fluorescent PCR, kuki igice cyagutse kigomba kugenzurwa murwego rwa 80-300bp?

Igisubizo.Igice cyibicuruzwa ni kigufi cyane kuburyo gitandukana na primer-dimer.Uburebure bwa primer-dimer ni 30-40bp, kandi biragoye gutandukanya niba ari primer-dimer cyangwa igicuruzwa niba kiri munsi ya 80bp.Niba igice cyibicuruzwa ari kirekire cyane, kirenze 300bp, bizoroha byoroshye gukora amplifisione nkeya kandi ntibishobora kumenya neza urugero rwa gene.

Kurugero, iyo ubaze umubare wabantu bari mwishuri, ugomba kubara gusa umunwa uhari.Ni nako bimeze iyo ubonye genes, ugomba gusa kumenya urutonde runaka rwa gene kugirango uhagararire Urutonde rwose ruzakora.Niba ushaka kubara abantu, ugomba kubara umunwa n'amazuru, amatwi, n'ibirahure, kandi biroroshye gukora amakosa.

Kwaguka, mubushakashatsi bwibinyabuzima, hariho ibibazo byinshi byubushakashatsi kuva aho bijya, kubera ko gene ikurikirana yubwoko ubwo aribwo bwose ari ndende cyane, ntibikenewe kandi ntibishoboka gupima ibice byose, urugero nka bagiteri 16S ikurikirana, aribyo gukora urutonde rwibintu bya bagiteri Isuzuma kugirango hamenyekane umubare wabaturage ba bagiteri.

Ikibazo: Ni ubuhe burebure bwiza bwo gushushanya primer ya qPCR?

Igisubizo: Mubisanzwe, uburebure bwa primer ni nka 20-24bp, nibyiza.Birumvikana, tugomba kwitondera agaciro ka TM agaciro ka primer mugihe dushushanya primer, kuko ibi bijyanye nubushyuhe bwiza bwa annealing.Nyuma yubushakashatsi bwinshi, byagaragaye ko 60 ° C nigiciro cyiza cya TM.Niba ubushyuhe bwa annealing buri hasi cyane, bizaganisha byoroshye kuri amplification idasanzwe.Niba ubushyuhe bwa annealing buri hejuru cyane, imikorere ya amplification izaba mike ugereranije, impinga yumurongo wa amplification izatangira nyuma, kandi agaciro ka CT kazatinda.

Ikibazo: Nigute uburyo bwo gusiga irangi butandukanye nuburyo bwa probe?

Igisubizo: Uburyo bwo gusiga irangiAmabara amwe ya fluorescent, nka SYBR Icyatsi Ⅰ, PicoGreen, BEBO, nibindi, ntibisohora urumuri rwonyine, ahubwo bizasohora fluorescence nyuma yo guhambira kumurongo muto wa ADN ebyiri.Kubwibyo, mugitangira reaction ya PCR, imashini ntishobora kumenya ibimenyetso bya fluorescent.Iyo reaction igeze kuri annealing-kwaguka, umugozi wikinguye urakingurwa, hanyuma umugozi mushya ugahuzwa mugikorwa cya ADN polymerase, kandi molekile ya fluorescent ihuza na dsDNA ntoya.Mugihe umubare wizunguruka za PCR wiyongera, amarangi menshi kandi menshi ahujwe na ADN ikubye kabiri, kandi ibimenyetso bya fluorescent nabyo bikomeza kwiyongera.Uburyo bwo gusiga irangi bukoreshwa cyane mubushakashatsi bwa siyansi.
PS: Witondere mugihe ukora igerageza, irangi rigomba guhuzwa na ADN yabantu, witondere kubihindura umuntu wa fluorescent.

Uburyo bwo gusiga irangi (ibumoso) Uburyo bwa Probe (iburyo)
PS: Witondere mugihe ukora igerageza, irangi rigomba guhuzwa na ADN yabantu, witondere kubihindura umuntu wa fluorescent.

SYBR Icyatsi Ⅰ ihuza na shobora ntoya ya ADN

Uburyo bwubushakashatsiTaqman probe niyo hydrolysis ikoreshwa cyane.Hano hari itsinda rya fluorescent kuri 5 ′ iherezo ryiperereza, mubisanzwe FAM, kandi iperereza ubwaryo ni urukurikirane rwuzuzanya na gen.Hano hari itsinda ryazimya fluorescent kuri 3 ′ iherezo.Ukurikije ihame ryo guhererekanya ingufu za fluorescence resonance (ihererekanyabubasha rya Förster resonance, FRET), mugihe itsinda ryumunyamakuru fluorescent (molekile ya donor fluorescent) hamwe nitsinda rya florescent rizimya (molekulaire ya fluorescent) ryishimiwe Iyo spekrice ihurira hamwe nintera iri hafi cyane (7-10nm), umunezero wa molekile ya donor irashobora kworohereza molekile ya fluorescence.Kubwibyo, mugitangira reaction ya PCR, mugihe iperereza ryubusa kandi ridahwitse muri sisitemu, itsinda ryumunyamakuru fluorescent ntirisohora fluorescence.Iyo annealing, primer na probe bihuza nicyitegererezo.Mugihe cyo kwagura, polymerase ikomeza guhuza iminyururu mishya.ADN polymerase ifite 5′-3 ′ ibikorwa bya exonuc Please.Iyo ugeze kuri probe, polymerase ya ADN izahindura hydrolyze iperereza nicyitegererezo, itandukanya itsinda ryumunyamakuru fluorescent nitsinda rya quencher fluorescent, kandi irekure ibimenyetso bya fluorescent.Kubera ko hari umubano umwe-umwe hagati yubushakashatsi hamwe nicyitegererezo, uburyo bwa probe buruta uburyo bwo gusiga irangi muburyo bwo kumenya no kumva neza ikizamini.Uburyo bwa probe bukoreshwa cyane mugusuzuma.

Ikibazo: Umubare wuzuye ni iki?Umubare ugereranije ni iki?

Igisubizo: Umubare wuzuye bivuga kubara umubare wambere wa kopi yintangarugero ugomba gupimwa na qPCR, nkanga virusi za HBV ziri muri 1ml yamaraso.Igisubizo cyabonetse kubigereranyo ugereranije nimpinduka mubunini bwa gen igenewe murugero rwihariye ugereranije nurundi rugero rwerekanwe, kandi imvugo ya gene iragenzurwa cyangwa igenzurwa.

Ikibazo: Ese ingano yo gukuramo RNA, guhinduranya inyandiko, hamwe no kongera imbaraga bizagira ingaruka kubisubizo byubushakashatsi?
Ikibazo: Ese icyitegererezo cyo kubika, gukuramo reagent, reagent transcription reagent, hamwe nibikoresho bikwirakwiza urumuri bizagira ingaruka kubisubizo byubushakashatsi?
Ikibazo: Ni ubuhe buryo bushobora gukosora amakuru yubushakashatsi?

Kubijyanye nibi bibazo, tuzabisobanura birambuye mubice byateye imbere kandi byateye imbere hepfo.
2. Ubumenyi buhanitse

Kubyerekeranye nigihe nyacyo cya fluorescent PCR, tugomba kumenya ukuri ko ibihumbi byubushakashatsi bwa siyanse bisohoka buri mwaka, muri byo tekinoroji ya PCR ya fluorescent ntabwo ari umubare muto.

Niba ntamahame asanzwe yo gupima igeragezwa rya fluorescent PCR, ibisubizo birashobora gutandukana cyane.Kuri gene imwe yubwoko bumwe, hamwe nuburyo bumwe bwo gutunganya, ibisubizo byo gutahura nabyo bizatandukana cyane, kandi bizagora abakererewe gusubiramo ibisubizo bimwe.Wowe Ntawe uzi igikwiye n'ikibi.

Ibi bivuze ko fluorescent quantitative PCR ari tekinoroji yibeshya cyangwa tekinoroji itizewe?Oya, ni ukubera ko PCR igereranya PCR irumva neza kandi neza, kandi imikorere itari mike izatanga ibisubizo bitandukanye rwose.Igihombo gito ni kilometero igihumbi.Umwanditsi wiyi ngingo arashobora kubabazwa inshuro nyinshi nababisubiramo.Mugihe kimwe, abasubiramo ikinyamakuru nabo biragoye guhitamo mubisubizo bitandukanye byubushakashatsi.

Byose muri byose, byerekana kubura ubwumvikane mugihe nyacyo cya PCR.Kugira ngo ibyo bishoboke, abahanga bakuru mu nganda batangiye gushyiraho ibipimo,gusaba abaterankunga gutanga amakuru akenewe yubushakashatsi no gutunganya amakuru (harimo amakuru akenewe) mu ngingo kugirango yuzuze ibipimo.

Abasesengura barashobora gusuzuma ireme ryikigereranyo basoma aya makuru;abasomyi b'ejo hazaza nabo barashobora gukoresha ibi kugirango basubiremo igerageza cyangwa kunoza igerageza.Noneho ibisubizo byubushakashatsi byabonetse murubu buryo byuzuye amakuru, ubuziranenge, kandi burakoreshwa.

MIBBI (Amakuru ntarengwa yiperereza ryibinyabuzima na Biomedical -http: // www.mibbi.org) yabayeho.MIBBI ni umushinga utanga ibipimo byubushakashatsi.Byatangajwe muri kamere.Uyu mushinga ugamije ubushakashatsi butandukanye bwibinyabuzima, harimo ibinyabuzima ngengabuzima, Microarray, qPCR tugiye kuganira ubu, nibindi, kandi butanga buri bwoko bwikigereranyo mugihe utanga inyandiko zandikishijwe intoki.Ayo makuru agomba gutangwa igihe cyose.

Mu mushinga wa MIBBI, hari ingingo ebyiri zijyanye na fluorescent quantitative PCR, aribyo:
· RDML (Igihe nyacyo PCR Data Markup Ururimi) - ururimi rwubatswe hamwe nigitabo cyo gutanga raporo kumibare nyayo ya PCR;
· MIQE (Amakuru ntarengwa yo gutangaza umubare wuzuye-Igihe cya PCR Ubushakashatsi) - amakuru ntarengwa yo gutangaza ingingo zerekeye igihe nyacyo cyo gupima PCR.
Ubwa mbere, reka tuvuge kuri RDML, ibisobanuro byamagambo.

Niba nta bisobanuro bisanzwe kuri buri kintu, ntibishoboka gukomeza ibiganiro, niyo mpamvu ibisobanuro byamagambo ari ngombwa mubizamini.
Ijambo ryakoreshejwe mukigereranyo cya fluorescent PCR ikubiyemo ibintu bikurikira.QIAGEN yakoze incamake nziza kuri twe.Ibikurikira byose byumyeibicuruzwa .

Amplification curve
Amplification curve yerekeza kumurongo wakozwe mugihe cya PCR, hamwe numubare wizunguruko nka abscissa hamwe nigihe nyacyo cya fluorescence mugihe cyo kubyitwaramo nkibisanzwe.

Inzira nziza ya amplification curve igomba kugira ibiranga bikurikira: ibyingenzi biringaniye cyangwa byagabanutseho gato, kandi nta cyerekezo kigaragara cyo kuzamuka;ihindagurika ry'umurongo urasobanutse, kandi ahahanamye icyiciro cya exponential iragereranywa no gukora neza.Nini ahantu hahanamye, niko ubushobozi bwo kongera imbaraga;muri rusange amplification curve Igereranya ni ryiza, byerekana ko imikorere ya amplification ya buri tube isa;icyiciro cya exponential ya amplification curve ya sample-concentration ntangarugero iragaragara.

Ibyingenzi (Baseline)
Ibyingenzi ni urusaku rwurwego rwambere, mubisanzwe bipimirwa hagati yukwezi kwa 3 na 15, kubera ko agaciro ka fluorescence katewe nigicuruzwa cyongerewe imbaraga ntigishobora kugaragara muriki gihe.Umubare wizunguruka zikoreshwa mukubara ibyingenzi zirashobora gutandukana kandi birashobora gukenera kugabanuka niba umubare munini wicyitegererezo ukoreshwa cyangwa niba imvugo urwego rwa gen igenewe ari ndende.

Gushiraho ibyingenzi bisaba kureba amakuru ya fluorescence uhereye kumurongo ugereranije amplification curve.Ibyingenzi byashyizweho kugirango imikurire ya amplification curve itangirana numubare wizenguruko uruta urwego rwibanze.Ibyingenzi bigomba gushyirwaho kugiti cya buri cyiciro gikurikiranye.Impuzandengo ya fluorescence yagaragaye mugihe cyambere cyambere igomba gukurwa mubiciro bya fluorescence byabonetse mubicuruzwa byongerewe.Ubusobanuro bwanyuma bwa software itandukanye-Igihe PCR itanga uburyo bwikora bwogutezimbere ibice byibanze byintangarugero.

Mugihe cyambere cyambere cya PCR amplification reaction, ibimenyetso bya fluorescence ntabwo bihinduka cyane.Kwegera umurongo ugororotse byitwa ibyingenzi, ariko iyo turebye neza ukuzenguruka kwambere, tubona ko muribanze aribyo bibera kumashusho hepfo.

Amavu n'amavuko
agaciro kadasanzwe ka fluorescence mubitekerezo.Kurugero: kuzimya fluorescence idakora neza;cyangwa umubare munini wa ADN yerekana inyandikorugero ebyiri kubera gukoresha SYBR Icyatsi.Ibice byinyuma byibimenyetso byavanyweho imibare na Real-Time PCR software algorithm.

Ikimenyetso cy'abanyamakuru
Ibimenyetso byabanyamakuru bivuga ibimenyetso bya fluorescente byakozwe na SYBR Icyatsi cyangwa fluoresc yanditseho ibimenyetso byihariye bikurikirana mugihe nyacyo PCR.

Ikimenyetso gisanzwe cy'abanyamakuru (RN)
RN bivuga ubukana bwa fluorescence bwirangi ryumunyamakuru ugabanijwe nuburemere bwa fluorescence yibara ryerekana irangi ryapimwe kuri buri cyiciro.

Irangi ryerekana
Mubihe bimwe-byukuri PCR,irangi rya fluorescent ROX ikoreshwa nkimbere yimbere kugirango isanzwe ibimenyetso bya fluorescent.Ikosora kubitandukanye kubera imiyoboro idahwitse, umwanya mwiza, hamwe nihindagurika rya fluorescence kumurongo mwiza-neza.

Inzira ya fluorescence (imbago)
yahinduwe hejuru yagaciro kinyuma kandi cyane munsi yibibaya bya plateau ya amplification curve.Igomba kuryama mukarere ka murongo wa amplification curve, byerekana log-umurongo urwego rwa PCR.Imipaka igomba gushyirwaho muburyo bwa log-amplification curve reba kugirango log-umurongo icyiciro cya PCR kimenyekane byoroshye.Niba hari genes nyinshi zigenewe muri Real-Time PCR, urwego rugomba gushyirwaho kuri buri ntego.Mubisanzwe, ibimenyetso bya fluorescence yibizunguruka 15 byambere bya reaction ya PCR bikoreshwa nkikimenyetso cyerekana inyuma ya fluorescence, kandi urwego rwa fluorescence rwikubye inshuro 10 gutandukana bisanzwe byerekana ibimenyetso bya fluorescence yikimenyetso cya 3 kugeza kuri 15 byambere bya PCR, kandi urwego rwa fluorescence rushyizwe mugice cyerekana ko PCR yongerewe imbaraga.Muri rusange, buri gikoresho gifite fluorescence ntarengwa yashyizweho mbere yo gukoreshwa.

Umuzenguruko wikiziga (CT) cyangwa Kwambukiranya (CP)
Umuzenguruko aho amplification curve yambukiranya imipaka (ni ukuvuga, aho fluorescence igaragara yiyongera cyane).CT irashobora kuba agace kandi ingano yo gutangira inyandikorugero irashobora kubarwa.Agaciro CT kerekana umubare wizunguruka zabayeho mugihe ikimenyetso cya fluorescent muri buri cyerekezo cya PCR kigeze kumurongo.Hariho umurongo ugaragara hagati ya CT agaciro ka buri nyandikorugero na logarithm yumubare wambere wimibare yicyitegererezo ,.hejuru umubare wambere wa kopi, ntoya CT agaciro, naho ubundi.Igipimo gisanzwe gishobora gukorwa ukoresheje igipimo gifite numero yambere ya kopi izwi, aho abcissa igereranya agaciro ka CT, naho ordinate igereranya logarithm yumubare wambere wa kopi.Kubwibyo, mugihe cyose CT agaciro ka sample itazwi yabonetse, nimero yambere ya kopi yicyitegererezo irashobora kubarwa uhereye kumurongo usanzwe.

ΔCT agaciro
ΔCT agaciro gasobanuraitandukaniro hagati yintego ya gen hamwe na endogenous reference gene CT agaciro, nka gene yo mu rugo, kandi ikoreshwa muguhuza urugero rwicyitegererezo cyakoreshejwe:
⇒ΔCT = CT (intego ya gene) - CT (endogenous reference gene)

ΔΔCT Agaciro
Agaciro ΔΔCT isobanura itandukaniro riri hagati ya ΔΔCT agaciro k'icyitegererezo cy'inyungu (urugero, ingirabuzimafatizo) hamwe n'agaciro ΔΔCT agaciro k'icyitegererezo (urugero, selile zidateganijwe).Icyitegererezo ntangarugero nacyo cyitwa kalibrasi ntangarugero kandi izindi ngero zose zisanzwe kuriyi kugirango ugereranye:
⇒ΔΔCT = impuzandengo ΔCT (icyitegererezo cy'inyungu) - impuzandengo ΔCT (icyitegererezo)

Endogenous reference genes (endogenous reference genes)
Urwego rwo kwerekana imiterere ya endogenous reference genes, nka gen zo kubungabunga urugo (genes zo mu rugo), ntizitandukanya ingero.Kugereranya indangagaciro za CT zerekana gene yerekanwe kugirango yemere urwego rwimiterere ya gene igenewe guhinduka muburyo bwo kwinjiza RNA cyangwa cDNA (reba igice kijyanye na valuesCT indangagaciro hejuru).

Imbere yimbere ikosora kuribirashoboka kwangirika kwa RNA cyangwa kuba hari inzitizi za enzyme mu ngero za RNA, kimwe no gutandukana mubirimo RNA, uburyo bwo kwandukura neza, kugarura aside nucleic, no gufata icyitegererezo.Guhitamo icyerekezo cyiza (s), twahinduye algorithm kugirango twemere guhitamo ibyatoranijwe neza biterwa nubushakashatsi.

Kugenzura imbere
Igenzura rikurikirana ryongerewe imbaraga muburyo bumwe nkurugero rwintego kandi bigakurikiranwa nubushakashatsi butandukanye (ni ukuvuga gukora duplex PCR).Igenzura ryimbere rikoreshwa kenshi kugirango wirinde kunanirwa kwongerewe, nkigihe intego ikurikiranye itamenyekanye.
Icyitegererezo
Icyitegererezo (urugero, RNA yatunganijwe kuva kumurongo wa selile cyangwa tissue) ikoreshwa mukigereranyo cyo kugereranya izindi ngero zose kugirango umenye urwego rwerekana imiterere ya gene.Calibration sample irashobora kuba icyitegererezo icyo aricyo cyose, ariko mubisanzwe ni igenzura (kurugero, icyitegererezo kitavuwe cyangwa icyitegererezo kuva igihe zeru cyubushakashatsi).

Kugenzura neza
Koresha uburyo bwo kugenzura hamwe naumubare uzwi w'icyitegererezo.Igenzura ryiza rikoreshwa kenshi mugusuzuma niba primer set cyangwa primer - probe set ikora neza kandi ko reaction yashyizweho neza.

Nta kugenzura icyitegererezo (NTC)
Igenzura ririmo ibintu byose bikenewe bya amplification reaction usibye inyandikorugero, ubusanzwe isimbuzwa amazi.Imikoreshereze ya NTC irashobora kubona umwanda uterwa no kwanduzwa kwa reagent cyangwa ADN y’amahanga, bityo bigatuma ukuri kwizerwa kwizerwa.Kwiyongera kugenzura NTC byerekana kwanduza.

Nta kugenzura RT (NRT)
Uburyo bwo gukuramo RNA bushobora kuba bukubiyemo ADN isigaranye ADN, yangiza cyane kandi niyo nyirabayazana igira ingaruka ku bwiza bw’amakuru ndetse n’umwanzi usanzwe wa qPCR, bityo rero mugihe utegura ubushakashatsi, ugomba kuba wateguwe kugirango wongere gusa RNA.Hariho inzira ebyiri, imwe ni ugushushanya primers hirya no hino, indi ni ugukuraho burundu ADN, imwe muribyiza, izaganirwaho nyuma.Igenzura rya NTR ni indorerwamo yubumaji kugirango hamenyekane umwanda wa ADN.Niba hari amplification, bivuze ko hariho umwanda.

Ibipimo
Ibipimo nicyitegererezo cyibizwi cyangwa kopi nimero ikoreshwa mukubaka umurongo usanzwe.Kugirango hamenyekane ituze ryurwego rusanzwe, igice cya gene gikunze gukoronizwa muri plasmid kandi kigakoreshwa nkibisanzwe.

Umurongo usanzwe
mubisanzwe bivangwa byibuze byibuze 5 yibikoresho hamwe nibicuruzwa bisanzwe ukurikije igipimo cyikubye kabiri, kandi amanota 5 yashushanijwe mumirongo ya CT agaciro na kopi nimero, kandi ingingo zahujwe no gukora umurongo kugirango habeho umurongo usanzwe.Kuri buri murongo usanzwe, agaciro kayo kagomba kugenzurwa.Agaciro kamanutse kagabanuka hagati ya -3.3 na –3.8, kandi buri kwibandaho bikorwa muri bitatu.Ingingo zitandukanye cyane nizindi ngingo zigomba gutabwa.Agaciro CT k'icyitegererezo kugeragezwa kazanwa mu murongo usanzwe, kandi imvugo y'icyitegererezo igomba kugeragezwa irashobora kubarwa.

CT agaciro k'icyitegererezo kugeragezwa kazanwa mumurongo usanzwe, kandi nimero yambere ya kopi yicyitegererezo igomba kugeragezwa irashobora kubarwa.

Gukora neza no kumurongo
Umusozi wumurongo usanzwe ugaragaza imikorere yigihe-nyacyo PCR.
· Umusozi wa -3.322 werekana ko PCR ikora neza ari 1, cyangwa 100%, kandi ibicuruzwa bya PCR byikuba kabiri kuri buri cyiciro.
· Umusozi uri munsi ya -3.322 (urugero, –3.8) werekana imikorere ya PCR
· Umusozi urenze –3.322 (urugero, –3.0) werekana ko imikorere ya PCR isa nkaho irenga 100%, bikaba amatsiko, nigute uruziga rumwe rwa PCR rwabyara inshuro zirenga ebyiri ibicuruzwa byongerewe?Ibi bintu bibaho mugice kitari umurongo wa reaction ya PCR, ni ukuvuga ko hari umubare munini wa amplification idasanzwe.

gushonga umurongo
Nyuma ya amplification ya qPCR irangiye, ibicuruzwa bya PCR birashyuha.Mugihe ubushyuhe buzamutse, ibicuruzwa byongerewe kabiri byongeweho buhoro buhoro bishonga, bigatuma ubukana bwa fluorescence bugabanuka.Iyo ubushyuhe runaka (Tm) bugeze, umubare munini wibicuruzwa bizashonga.Fluorescence igabanuka cyane.Ibicuruzwa bitandukanye bya PCR bifite agaciro ka Tm nubushyuhe butandukanye bwo gushonga, kugirango umwihariko wa PCR ubashe kumenyekana.

Gushonga umurongo (derivative curve)
Gushonga umurongo biva muburyo bwo gukora ikarita yimpinga, irashobora kwerekana byimazeyo imiterere yibicuruzwa bya PCR.Kubera ko ubushyuhe bwo gushonga ari agaciro ka Tm k'igice cya ADN, ibipimo bimwe na bimwe bigira ingaruka ku gaciro ka Tm k'igice cya ADN birashobora gucirwa urubanza, nk'ubunini bw'ibice, ibirimo GC, n'ibindi. Muri rusange, dukurikije amahame yacu yo gushushanya,uburebure bwibicuruzwa byongerewe imbaraga biri hagati ya 80-300bp, bityo ubushyuhe bwo gushonga bugomba kuba hagati ya 80 ° C na 90 ° C.

Ibisobanuro byo gushonga umurongo: Niba impinga nyamukuru yonyine igaragara hagati ya 80 ° C-90 ° C, bivuze ko PCR igereranya PCR itunganye;niba impinga nyamukuru igaragara hagati ya 80 ° C-90 ° C naho impinga zitandukanye zigaragara munsi ya 80 ° C, primer dimer irasuzumwa.Urashobora kugerageza kongera ubushyuhe bwa annealing kugirango ubikemure;niba impinga nyamukuru igaragara hagati ya 80 ° C-90 ° C, naho impinga zinyuranye zikongera kugaragara mugihe ubushyuhe buzamutse, muri rusange bifatwa ko hariho ADN yanduye, kandi ADN igomba gukurwaho mugihe cyambere cyubushakashatsi.

Birumvikana ko haracyari ibintu bidasanzwe, bizacika umwe umwe hepfo.
3. Ubumenyi buhanitse

Gukora qPCR, ngomba kuvuga MIQE,Amakuru Ntarengwayo gutangazaUmubareIgihe nyacyo PCRUbushakashatsi - amakuru make yo gutangaza ingingo zerekeye igihe nyacyo PCRubushakashatsi.Kugirango tworoshe imyumvire ya buri wese, tuzoroshya ibyingenzi.

Urashobora gushakisha inyandiko yumwimerere ya MIQE kuri enterineti, kandi icyingenzi nuko iteganyaurutonde rwamakuru rugomba gutangwa mugihe utangaza ingingo .

Abasesengura barashobora gusuzuma ireme ryikigereranyo basoma aya makuru;abasomyi b'ejo hazaza nabo barashobora gukoresha ibi gusubiramo cyangwa kunoza igerageza.
Birakwiye ko tumenya ko mururu rutonde, akamaro ka buri rutonde kagaragajwe na E cyangwa D.Bisobanura iki?E: amakuru yingenzi (agomba gutangwa);D: amakuru yifuzwa (tanga byinshi bishoboka).

MIQE (1) - Igishushanyo mbonera
Abatekamutwe benshi barangije kwirwanaho nyuma yo kurangiza amasomo yabo ntibazamenya gukora igerageza ryigenga, gufungura amakaye yabo, no gukora ibyo mwarimu ababwiye gukora.Kubera iyo mpamvu, igishushanyo mbonera nticyari gikomeye, kandi ishami ryandika ryikinyamakuru rivuga ko bashaka gukora iyi shusho niyi shusho, nuko babikora batangaye.Uku nuburyo ibisebe bikozwe!

Hafi y'urugo, ihame rya mbere ryikigereranyo ni ukumenyagukomera kwa logique yubushakashatsi.Ikintu cyibanze cyane ni igishushanyo mbonera, kandi ikintu cyingenzi kijyanye nigishushanyo mbonera ni uburyo bwo gushyiraho icyitegererezo, icyitegererezo (kugenzura), n'umubare wabisubiramo, kugirango amakuru yubushakashatsi ashobore kwerekanwa, kugereranywa, no kwemeza.

Icyitegererezobivuga icyitegererezo kidusaba kumenya gene igenewe nyuma yo kuvurwa runaka.Icyitegererezoni icyitegererezo nta buryo bwo kuvura, bukunze kwitwa ubwoko bw'ishyamba muri biologiya.

Ubushakashatsi bwiganani ngombwa cyane.Mubisanzwe, umubare wigana wigana ugomba kuba urenze itatu.Birakenewe gutandukanya icyo kwigana ibinyabuzima nicyo kwigana tekiniki.

Ibinyabuzima bigana: Ikigeragezo kimwe cyo kugenzura cyakozwe hamwe nibikoresho bitandukanye (igihe, ibimera, ibyiciro, ibyapa byerekana).

Kwigana ibinyabuzima
Reka dufate urugero rwumuti wica udukoko twa pepper.Turashaka gutera imiti yica udukoko ku bimera bitatu bya ABC, hanyuma ibihingwa bitatu bya ABC ni bitatu byigana ibinyabuzima, kandi ni ubushakashatsi bumwe bwo kugenzura bwakozwe nibikoresho bitandukanye.Ariko nkubushakashatsi, birakenewe rwose kugenzura, kuburyo dushobora gutera rimwe mumashami yikimera A kugirango dushyireho itsinda ryikigereranyo cyibimera A, kandi ntitutere andi mashami yikimera A kugirango dushyireho itsinda rishinzwe kugenzura.Kora kimwe kuri B na C.

Ubuhanga bwa tekinike (Replicates tekinike): Nubushakashatsi bwasubiwemo bugamije kwirinda amakosa yatewe nigikorwa, mubyukuri ni umwobo wikopi ushyizwe mubintu bimwe.Ubuvuzi hamwe nubugenzuzi byombi bigomba kuba byigana igenamigambi (byibuze bitatu) bya gen igenewe na gen imbere.

Gusubiramo tekinike
Fata urusenda ruvurwa nudukoko twangiza urugero.Kubitsinda ryubushakashatsi bwibimera A, twakoze imyobo itatu ya PCR ya 1, 2, na 3 kuri gen igenewe hamwe na gene yerekanwe imbere, kugirango dufate ikigereranyo nyuma yo gutahura.Kugenzura ibimera Amatsinda nayo afatwa muburyo bumwe.Mu buryo nk'ubwo, kora ubuvuzi bumwe kubihingwa B na C.Ubu ni gusubiramo tekinike.

Birakwiye ko tumenyaicyinjira mu mibare ni ugusubiramo ibinyabuzima, kandi gusubiramo tekiniki ni ukugerageza kumenya niba hari ibintu bitunguranye muburyo bwo kugerageza, kugirango ibisubizo byubushakashatsi byizewe, ni ukuvuga kwirinda amakosa ufata impuzandengo yabo nkuko dukunze kubivuga.

Igenzura ribi - NTC na NRT
NTC (Nta-Igenzura), igenzura ridafite inyandikorugero, rikoreshwa mukugenzura niba ibikoresho byubushakashatsi byanduye.Mubisanzwe, amazi akoreshwa nkicyitegererezo.Niba hari reaction ya fluorescent, yerekana ko kwanduza aside nucleic byabaye muri laboratoire.

Iyi myanda iva: amazi yanduye, reagent zujuje ibyangombwa zirimo ADN ya endogenous, kwanduza primer, kwanduza ibikoresho bya laboratoire, kwanduza aerosol, nibindi, bigomba gukoresha ibyuma bya RNase hamwe na inhibitori ya RNase.Guhumanya ikirere nikigoye kubibona.Tekereza laboratoire yawe imeze nkumwotsi, hamwe na acide nucleic zitandukanye zahagaritswe mukirere.

NRT (No-Reverse Transcriptase), igenzura ridafite transcript, ni RNA idasubirwaho yandukuwe nka RNA igenzura nabi, ikaba igenzura ibisigisigi bya gDNA.

Iyo ukora imvugo ya gene, ingano ya RNA igaragazwa no kumenya ingano ya cDNA nyuma yo kwandukura.Niba hari ibisigazwa bya gDNA mugihe RNA isukuwe, bizatera amakosa mubisubizo byubushakashatsi, kuko ibisubizo nyabyo byabonetse ni gDNA na cDNA.Kurwego rusange, ntabwo ari cDNA gusa, gDNA igomba gukurwaho burundu mugihe cyo gukuramo RNA.

MIQE (2) - amakuru yintangarugero
Ibyo bita sample yamakuru bisobanura ko mugihe dutangaje inkuru ivuga kuri qPCR, tugomba gusobanura neza amakuru yintangarugero, nikintu cyingenzi mubiganiro.Mu buryo nk'ubwo, mugihe dutunganya ibyitegererezo, tugomba no kugenzura ibikorwa byacu kugirango tumenye neza ibyitegererezo.

Ibisobanuro by'icyitegererezo ni ibisubizo gusa, kandi tugomba kurushaho kwita kubikoresho byafashwe mugihe cyose cy'igerageza.

Guhitamo ibikoresho byubushakashatsi
Ingero zamaraso - hitamo amaraso mashya, bitarenze amasaha 4.Ingero z'utugari - hitamo gukusanya selile nshya mugihe cyo gukura gukomeye.Inyama zinyamanswa - Hitamo ibice bishya, bikura cyane.Tissue Tissue - Hitamo imyenda mishya, ikiri nto.

Ugomba kuba wabonye ko hari ijambo ryingenzi muriyi nteruro nke: shyashya.
Kuburugero rwavuzwe haruguru, ibikoresho byiza, bidahenze, kandi bihamye kumasoko nigikoresho cya Foregene, gishobora gukuramo vuba kandi byoroshye ADN na RNA.

Amaraso ADN Mini Kit

Akagari Igiteranyo cya RNA Igikoresho cyo kwigunga

Inyamaswa Igiteranyo Cyuzuye RNA

Tera Igiteranyo Cyuzuye cya RNA

Tera Igiteranyo Cyuzuye cya RNA Kwigunga

Tera ibikoresho bya ADN byo kwigunga

Kubika ibikoresho byubushakashatsi
Muri rusange, ntabwo dushaka kubika ingero, niba ibintu byemewe.Nyamara, hari inshuti nyinshi zidashobora gukora ubushakashatsi ako kanya nyuma yo gutoranywa, ndetse bamwe bakeneye no gutwara ibigega bya azote byamazi mumurima kugirango babitange.

Kubwubu bwoko bwinshuti ikora cyane, ndashobora kuvuga gusa ko udasobanukiwe nibishobora gukoreshwa.Ubu ibigo byinshi byongera gukoresha reagent bitanga reagent zishobora kubika ibyitegererezo bya RNA mubushyuhe bwicyumba, kandi ushobora guhitamo kubikoresha.Uburyo busanzwe bwo kubika ni ububiko bwa azote, ukoresheje ikigega gito cya azote cyoroshye gutwara.Nyuma yo kugarura icyitegererezo muri laboratoire, ubibike muri firigo -80 ° C.

Kubushakashatsi burimo RNA, ihame ryamagambo atandatu rigomba gukurikizwa:ubushyuhe buke, nta enzymes,nabyihuse .

Igitekerezo cyubushyuhe buke biroroshye kubyumva;udafite enzymes, RNase iri hose kwisi dutuye (bitabaye ibyo waba warishwe na virusi itera sida), ubwo rero wakwirinda RNase mugihe ukora ubushakashatsi nigitekerezo gikomeye;byihuse,Nta Kung Fu ku isi idashobora gucika, gusa umuvuduko ntushobora gucika.

Kubwibyo, muburyo bumwe, igihe gito cyo gukuramo, nibyiza nibikoresho.KukiForegene'ibikoresho byibanda ku muvuduko, kuko babizi neza.

PS: Abakobwa bamwe bakora ubushakashatsi bitonze, ariko ntabwo bameze neza nka slam dunk nyuma yimyaka myinshi yakazi.Bumva ko Imana irenganya, kwinubira abandi, no gushaka ubuzima.Mubyukuri, ntabwo yabisobanukiwe.Ntabwo yarinze RNA neza, kandi umukinnyi wa slam dunk yari afite ubwenge.Igihe yakoraga igerageza, yatekereje ko azarangiza dlam dunk inshuro eshatu, inshuro eshanu n'amacakubiri abiri, ariko yakoze ubushakashatsi neza.

Icyitonderwa: Buhoro, amahirwe menshi yo gutera RNase.Nigute ushobora kwitoza kwihuta?Nta kuntu, kora imyitozo myinshi.

Kubushakashatsi butandukanye hamwe nicyitegererezo gitandukanye, biracyakenewe gusoma ibitabo byinshi no guhitamo uburyo bukwiye bwo gutunganya.Kuburyo bwo gukusanya no kubika icyitegererezo, MIQE isaba ko igomba kuba yanditse neza mu mpapuro, kugirango abasuzuma basuzume ubwizerwe bwimpapuro, kandi biranoroheye abakiri bato batangaye gusubiramo ubushakashatsi bwawe.

Nubwo ubushakashatsi bwibinyabuzima bugoye, burangije-hejuru.Niba utitonze, urashobora guhirika isi.Kurugero, gukora SARS mubibazo byibinyabuzima, cyangwa gukora umuceri wa Hybrid kugirango ukize abantu miliyari 1,3.Ishusho hepfo nubushakashatsi bwa chimique, ugomba kumva uburyo wishimiye ubushakashatsi bwawe ukareba gusa isura ye isa.Wibagirwe, ntukamwirabura.

MIQE (3) - gukuramo aside nucleic.
Gukuramo aside nucleique nikintu kinini, kandi ubushakashatsi bwibinyabuzima byose bya molekuline butangirana no gukuramo aside nucleic.Mbere ya byose, reka twandukure ibiri muri MIQE kubijyanye no gukuramo aside nucleic.

Urebye iyi fomu, ntushobora kuguma hejuru.Imiterere ni dogma.Kugirango ube umunyeshuri wambere, ugomba kubaza impamvu.Ibyingenzi bikubiye muri iyi mbonerahamwe ni: Gukurikiranaubuziranenge, ubunyangamugayo, guhuzagurika, no gukuramo RNA .

Igice cya mbere cyainzira cyangwa igikoresho nintambwe yo gukuramo aside nucleic.Niba ukoresha acide nucleic acide ikuramo kugirango ikuremo (yateye imbere, nyamuneka andikira kugura), ugomba kwerekana izina ryicyitegererezo cyigikoresho.

Izina ryigikoresho na

ni ikihe gikoresho cyakoreshejwe muburyo bwo guhindura ibisobanuro, reagent zidasanzwe zongeweho cyangwa nibikorwa bidasanzwe byakozwe bigomba gusobanurwa neza kugirango abandi basubiremo byoroshye ubushakashatsi bwawe.

Abantu bamwe bongeraho reagent zidasanzwe mugihe bakuramo ingero zidasanzwe, bibwira ko arintwaro yabo y'ibanga kandi ntibabwire abandi.Mugihe ubigira ibanga, banabura amahirwe yo gukora ingingo yawe.Ntukabe umunyabwenge, ugomba kuba inyangamugayo kuruta igihugu cya Zhang umusaza mubushakashatsi bwa siyanse, niba ushaka kuba umunyabwenge, ingingo izakugira umuswa.

ugomba kwibuka ibicuruzwa byumubare wibikoreshomugihe utumije ibikoresho hanyuma wandike ingingo.Muri rusange hariho imibare ibiri kuriki gikoresho: Injangwe - numero ya catalog (numero yibicuruzwa, nimero yingingo), Lot - nimero yibicuruzwa (Byakoreshejwe kwerekana icyiciro ibicuruzwa byaturutse).

Mubyongeyeho, nimero ya CAS ikoreshwa kenshi mugutumiza ibinyabuzima bya biohimiki, kandi nzabimenyekanisha hamwe.Umubare CAS numubare watanzwe na societe yimiti yabanyamerika kuri buri muti mushya wimiti.Mubisanzwe, imibare itatu ihujwe numurongo.CAS nomero ya Rushui: 7732-18-5.Imiti ikunze kugira amazina menshi, ariko numero CAS irihariye.Mugihe utumiza imiti, urashobora kubanza gusuzuma nimero ya CAS.

Hafi y'urugo, kuki tugomba gusobanura neza ibi bintu?Mubyukuri, ni no kugenzura ubuziranenge bwo gukuramo RNA.Gukoresha ibikoresho nibikoresho bizatuma gukuramo RNA bihoraho.Igipimo cyo gukuramo laboratoire zisanzwe ntabwo ari kinini, kandi gishobora kuboneka hamwe nibikoresho.

Ibisobanuro byo kuvura DNase cyangwa RNase
Ikibazo cyingenzi cya PCR igereranya PCR ni ukurinda kwanduza ADN, kandi ntugerageze niba hari umwanda.Niyo mpamvu, ni ngombwa kuvuga inzira wakoresheje mugutunganya ADN, kugirango werekane ko ADN mubikorwa byubushakashatsi yakuweho burundu.ihagarariwe nigishushanyo mbonera.

Igishushanyo mbonera cya RNA na ADN
Muri rusange, uburyo bwo gukuraho ADN ni ukuvura RNA hamwe na DNase nyuma yo kuyikuramo.Nyamara, ubu ni uburyo bukera.Ibikoresho byo gukuramo RNA byubucuruzi byashoboye gukuraho ADN mugihe cyo kuyikuramo utongeyeho DNase.Kurugero, urukurikirane rwibikoresho kuva Foregene.

Icyitonderwa: Gukuraho ADN mugihe cyo gukuramo RNA ninkota iteye ubwoba cyane yinkota ebyiri, izongera igihe cyo gukora cyo gukuramo RNA kandi byongere ibyago byo kwangirika kwa RNA.Ahanini, ni ubucuruzi hagati yumusaruro wa RNA nubuziranenge.

Mubyongeyeho, ingano ya DNase yongewe kuri silika ishingiye kuri adsorption inkingi ni nto cyane, kandi DNase yo mu rwego rwo hejuru igomba gukoreshwa kugirango igere ku ngaruka.DNase idakoreshwa ntishobora gusya vuba kandi byuzuye.Iki nikizamini cyurwego rwa tekiniki rwumucuruzi.Nibyo, hari nabandi bacuruzi badasanzwe birata ko ADN ishobora gukurwaho nta DNase.Birashobora kuvugwa ko umuntu wese wirata ko ADN ashobora gukurwaho burundu nta DNase ni hooligan.ADN isa naho ihagaze neza, kandi ntishobora guhanagurwa no kuvuga no guseka.

Isuzuma ryanduye
uburyo bwo gusuzuma: gutahura electrophoreis, 1% agarose, 6V / cm, 15min, gupakira 1-3 ul

Isesengura ryinshi rya acide nucleique
ni bisanzwe bipimwa ukoresheje UV yerekanwe.Reka mbanze menye ibisobanuro byindangagaciro eshatu za OD260, OD280, na OD230.
· OD260nm: Nuburebure bwumurambararo wimisemburo mwinshi wa acide nucleic, kandi agaciro gapimwe neza kuva kuri 0.1 kugeza 1.0.Niba atariyo, shyira cyangwa ushireho icyitegererezo kugirango uzane murwego.
· OD280nm: Nuburebure bwumurambararo wimisozi mwinshi wa proteine ​​nibintu bya fenolike.
· OD230nm: Nuburebure bwumurambararo wimisozi mwinshi wa karubone.

Ibikurikira, reka tuvuge ku ruhare rwa buri kimenyetso.Kuri A260, irashobora gukoreshwa mugupima umusaruro wa acide nucleic.Iyo OD260 = 1, dsDNA = 50μg / ml, ssDNA = 37μg / ml, RNA = 40μg / ml.

Kugira isuku, dukeneye kureba ibipimo dusanzwe tubona: OD260 / 280 na OD260 / 230.
· ADN yuzuye: OD260 / 280 ihwanye na 1.8.Iyo irenze 1.9, byerekana ko hariho umwanda wa RNA, kandi iyo uri munsi ya 1.6, byerekana ko hariho umwanda wa poroteyine na fenol.
· RNA yuzuye: 1.7
· OD260 / 230: Yaba ADN cyangwa RNA, agaciro kerekana ni 2.5.Iyo itageze kuri 2.0, byerekana ko hariho umwanda wisukari, umunyu nibintu kama.

Ubunyangamugayo bwa RNA

Ni ngombwa cyane gupima ubusugire bwa RNA.Mubisanzwe, birakenewe gukora igeragezwa rya RNA denaturation gel kugirango tumenye niba umucyo uri hagati ya 28S na 18S RNA ari umubano wikubye kabiri.Iyo itsinda rya gatatu 5S rigaragaye, bivuze ko RNA yatangiye kwangirika, usibye inyamaswa zidafite ubuzima.

Amakuru yo gusuzuma ubuziranenge bwa RNA: Usibye ibizamini byavuzwe haruguru, hari n'ibindi bikoresho byateye imbere mu bijyanye n'ubusugire bwa RNA, nk'ikizamini cya RQI cyuzuye cya sisitemu ya Experion automatic electrophoresis sisitemu, ishobora kumenya niba RNA yangiritse ku buryo butagaragara.

Mu bushakashatsi bwa siyansi, PCR igereranya PCR ni igereranya hagati ya gen igenewe na gen imbere.Kubwibyo, murwego rwo kubungabunga icyitegererezo cya RNA, gukuramo RNA, nibindi, intego yibanze nukureba ubusugire bwa RNA.

Ukuntu ubunyangamugayo bwa RNA bugira ingaruka ku buringanire hagati ya gen igenewe na gene yimbere irashobora kumvikana byoroshye uhereye kumashusho hepfo.Gutesha agaciro bizaganisha kuri gene ituzuye, yaba ituzuye ya gene yimbere yimbere cyangwa ituzuye ya gen igenewe, bizagira ingaruka zikomeye kumibare.

Igishushanyo mbonera cya gene igenewe na gene yerekanwe, ntigomba kuba impamo

Ikizamini cyo kubuza (niba agaciro ka CT karahagaritswe murwego rwo hejuru cyangwa ruto cyangwa ibindi bintu)

Dufashe iyi shusho nkurugero, Ct indangagaciro yimirongo itanu niyi ikurikira.Ikwirakwizwa rya CT indangagaciro hagati yumurongo ntiruringaniye, kandi indangagaciro za Ct ziratinda munsi yibitekerezo byinshi kandi bike, aribyo bibuza PCR kubuza.

Ingingo y'ingenzi: Muburyo bwo gukuramo RNA, dukeneye kureka imyumvire itari yo no gushyiraho ibikwiye.

Igitekerezo kitari cyo ni: Gukuramo RNA bikurikirana gusa umusaruro, utekereza ko uko umubare wa RNA wabonye ari mwiza.Mubyukuri, iyo dukora quantification, niba umubare wa gen utari munini cyane, ntidukeneye RNA cyane.Ingano ya RNA ukuramo irarenze bihagije.

Igitekerezo gikwiye ni:Gukuramo RNA bigomba gukurikirana ubuziranenge, ubunyangamugayo no guhuzagurika.Isuku irashobora kwemeza ko iyandikwa ryakurikiyeho ridahagarikwa kandi amakuru ntazagerwaho na ADN.Ubunyangamugayo butanga uburinganire bwintego zikurikirana hamwe nimbere.Guhoraho bitanga icyitegererezo gihamye.

MIQE (4) - guhinduranya inyandiko
Imyumvire mibi: gukurikirana urugero rwinshi rwicyitegererezo.
Igitekerezo gikwiye: Kurikirana ubudahwema (stabilite), utitaye ku mubare wa RNA yapakiwe, imikorere yo kwandukura inyandiko ikomeza kuba ihamye, kwemeza ko itandukaniro muri cDNA rishobora kwerekana itandukaniro muri mRNA.
Turasobanura iyi nzira hamwe nigishushanyo mbonera:

Igishushanyo mbonera cyerekana uburyo bwo guhindura inyandiko, ntukabe impamo
Mbere ya byose, dukeneye gusobanukirwa itandukaniro riri hagati yuburyo bwo kwandukura inzira hamwe na PCR.PCR ikora ubushyuhe bwinshi hamwe na annealing, kandi igice cyintego gikura cyane;mugihe transcription transcription idafite iyi nzira, turashobora kwiyumvisha ko transcript transcript mubyukuri mubyukuri umwe-umwe Mugihe cyo kwigana, nkibice byinshi bya RNA

nkuko haribishobora kubona ibice byinshi bya cDNA Amakuru, bigomba kumvikana nonaha, kuko ibice binini na bito byahinduwe-byandukuwe, kandi ntibishoboka kwibanda ku gice kimwe.Kandi kubera ko ingano ya RNA ari mike, ingano ya cDNA yabonetse nayo ni ntoya, bitandukanye na PCR, ifite ingaruka zo kongera imbaraga, kubwibyo ntibishoboka kubimenya.

cDNA ibisubizo bya electrophoreis
Icya kabiri, nibyiza, transcription transcription ikorwa umwe-umwe, ariko nta transcript transcript yavuye mubisosiyete iyo ariyo yose ishobora kugera kuriyi ngaruka.Ahanini, imikorere yinyandiko-mvugo zinyuranye zizerera hagati ya 30-50%.Niba aribyo, twahitamo kugira imikorere ihindagurika ihindagurika, aribyo dushaka kubona kuri iki gishushanyo: 3 RNA ibona cDNA 2, 6 RNAs ibona cDNA 4, niyo rero urugero rwaba rwaremerewe gute, uburyo bwo guhindura inyandiko burahinduka.Ntabwo dushaka kubona aho imikorere yo kwandukura ihindagurika idahungabana kandi kwibanda cyane.

None, nigute ushobora kugenzura niba imikorere yinyandiko mvugo ihagaze neza?Uburyo buroroshye cyane, ukeneye gukora ikigereranyo cyo kugereranya: kimwe ni uguhindura kwandukura muri cDNA nyuma yo gukuba kabiri kwa RNA, naho ubundi ni ugukora inshuro ebyiri nyuma yo kwandukura muri cDNA, hanyuma ugakora qPCR kugirango urebe ahahanamye Ese birahuye.Nkumunyeshuri wambere, ugomba kubyumva mumasegonda.Nkuko bigaragara hano hepfo:

Gukoresha RNA na cDNA kugirango ugerageze niba imikorere yo kwandukura ihagaze neza
Hindura inyandiko-mvugo n'ibikoresho
Nigute ushobora gutunganya neza fluorescent PCR ifite verisiyo nziza ya transcriptase hamwe nibikoresho.Guhindura inyandiko-mvugo igabanijwemo ubwoko bubiri ukurikije isoko, AMV cyangwaM-MLV, kandi imikorere yabo ni nkiyerekanwa kumeza.

Igikorwa cya RNase H.
RNase H ni Ribonuclease H, izina ryigishinwa ni ribonuclease H, ni endoribonuclease ishobora cyane cyane hydrolyze RNA mumurongo wa ADN-RNA.RNase H ntishobora hydrolyze ya fosifisiyumu ihuza ADN imwe cyangwa RNA imwe, cyangwa RNA, ni ukuvuga ko idashobora gusya ADN imwe cyangwa RNA imwe.Bikunze gukoreshwa muri synthesis yumurongo wa kabiri wa cDNA.

Ni ikintu kidasanzwe.Turavuga ko transcript transcript ifite ibikorwa bya RNase H, ntabwo bivuze ko transcriptase irimo RNase H, kandi ntibishoboka gutandukanya RNase H na transcriptase, wenda kubera ihinduka ryamatsinda amwe mumyandikire yinyuma Iki gikorwa giterwa na transcriptase.

Kubwibyo, tutitaye kumikorere ihanitse yo kwandukura ya AMV, ibikorwa byayo RNase H bigabanya umusaruro wa cDNA.Nibyo, abakora reagent bahora batezimbere ibicuruzwa byabo kugirango bakureho ibikorwa bya RNase H muri transcriptase ishoboka kugirango bongere umusaruro wa cDNA.
Ubushyuhe bwa Annealing

Icyiciro cya kabiri cya RNA mubushyuhe butandukanye
Reba igishushanyo hejuru hejuru yuburyo bwa kabiri bwa RNA mubushyuhe butandukanye, hanyuma ukoreshe igikoresho cya mFold kumurongo kugirango umenye imiterere ya kabiri yibice byateganijwe mugihe cy'ubushyuhe bwihariye hamwe nubushyuhe bwumunyu.Kuri 55 ° C, imiterere ya kabiri ya RNA iracyari ingorabahizi, transcriptase ntishobora gukora, kandi imiterere ya kabiri ntishobora gukemurwa burundu kugeza kuri 65 ° C, mugihe ubushyuhe bwiza bwa AMV na M-MLV buri munsi yubushyuhe.
gukora iki?Imiterere ya kabiri niyuzuzanya ryicyitegererezo ubwacyo, biganisha kumarushanwa akomeye hagati ya primer na reverse transcriptase hamwe nicyitegererezo, bikavamo urukurikirane rwibibazo nka E nkeya no gusubiramo nabi.

gukora iki?Gusa ongera ubushyuhe bwa annealing bishoboka.

Abenshi mubakora reagent barimo kunoza inyandiko zinyuranye binyuze mubwubatsi.Bamwe bongera ubushyuhe bwa reaction, nka Jifan na Aidelai, ndetse bamwe bakuraho itsinda rikora rya enzyme ya RNase H kugirango banoze isano iri hagati ya enzyme nicyitegererezo cya RNA.Kwiyegereza cyane birashobora guhatanira gusibanganya urwego rwa kabiri hanyuma ugasoma neza, kandi bikanatezimbere cyane imikorere ya transcript.
Ingingo y'ingenzi: Kwandukura kwinyuma ni ngombwa cyane kugirango ukurikirane uburyo bwo kwandukura neza (enzymes ntizigomba gukora neza gusa ahubwo zihamye), aho kuba urugero rwicyitegererezo cyapimwe, niba atari runini runini rwa fluorescent PCR, ntabwo bizashoboka na gato.CDNA nyinshi.
Inganda zinyuranye nazo zashyizeho ingufu mugukurikirana ubudahwema.Kurugero, ibigo byinshi ubu byapakiye inyandiko zinyuranye nkigikoresho gisanzwe cyo kugurisha, nikintu cyiza.
Kurugero, Foregene ya RT Yoroshye Ibikoresho:

RT Byoroshye I (Master premix kumurongo wambere cDNA synthesis kit)

MIQE (5) - amakuru ya gen

Igishushanyo cyavuzwe haruguru kirasobanura
1. Niba iyi gen ifite akamaro mubushakashatsi bwisubiramo birashobora kugenzurwa nubushakashatsi bwakorewe.
2. Indangamuntu ya Gene, urabizi.
3. Uburebure bwa gen, uburebure bwuzuye bwa gen igenewe rwose ntakibazo.Mugihe utegura primers, menya neza ko uburebure bwa amplicon buri hagati ya 80-200bp kugirango umenye neza imikorere ya amplification.
4. Urukurikirane rwo gutondekanya amakuru yo kugereranya, gene igenewe igomba kugereranwa muri genebank kugirango wirinde amplification idasanzwe.
5. Kubaho kwa pseudogene.Pseudogene ni urutonde rwa ADN rusa na gene isanzwe ariko itakaza imikorere isanzwe.Bikunze kubaho mumiryango myinshi ya eukaryote.Ubusanzwe ihagarariwe na ψ.Ni kopi ya ADN idakorwa muri genome isa cyane na code ya gen ikurikirana., muri rusange ntabwo yandukuwe, kandi nta busobanuro busobanutse bwa physiologique.
6. Umwanya wa primers ugereranije na exons na introns.Mu myaka ya mbere, igihe twakemuraga ikibazo cyo kwanduza ADN, twakunze kwita ku myanya ya primers, exons, na enteron, kandi muri rusange twatekerezaga gushushanya primers hirya no hino kugirango twirinde kwaguka kwa ADN.Nyamuneka reba igishushanyo gikurikira: umukara ugereranya inyuguti, ubururu butandukanye bugereranya exons, umutuku ugereranya primers isanzwe, naho umutuku ugaragara ugereranya intangiriro-izenguruka primers.

Igishushanyo, ntabwo ari ukuri
Mbega gahunda nziza ibi bisa, ariko mubyukuri, mubihe byinshi, trans-intron primers ntabwo ari amarozi nkuko wabitekerezaga, kandi bizanatera amplification idasanzwe.Inzira nziza rero yo kwirinda kwanduza ADN ni ugukuraho ADN burundu.
7. Guhanura guhinduka.Koresha uru rugero nanone, koresha igikoresho cya mFold kumurongo kugirango umenye imiterere ya kabiri yibice bigenewe ubushyuhe bwihariye hamwe nubunyu bwumunyu.

Icyiciro cya kabiri cya RNA mubushyuhe butandukanye
Imiterere ya kabiri niyuzuzanya ryicyitegererezo ubwacyo, bizaganisha kumarushanwa akomeye hagati ya primer na template, kandi amahirwe yo guhuza primer ni make, bikavamo urukurikirane rwibibazo nka E nkeya no gusubiramo nabi.Binyuze mu guhanura software, niba nta kibazo cyimiterere ya kabiri, byaba byiza.Niba bihari, ingingo yacu yo gukurikirana izaganira byumwihariko uburyo bwo gukemura iki kibazo.

MIQE (6) —qPCR Oligonucleotide

Kuri fluorescent quantitative PCR, ikintu cya mbere urwana na burimunsi ni ugukuramo RNA, kandi ikintu cya kabiri gishobora kuba igishushanyo mbonera.
Mbere ya byose, turacyagenzura amategeko yerekeye igishushanyo mbonera dukurikije urutonde rwa MIQE.Nibyoroshye cyane kuburyo ibisebe bishobora guseka, kandi dushobora kubirangiza mu nteruro imwe: shakisha uko bikurikirana hamwe numwanya wa primer probe nuburyo bwo guhindura.Kuburyo bwo kweza primer, synthesis ya primer ihendutse cyane kurubu, qPCR ikwiye PAGE no hejuru yuburyo bwo kweza, kandi amakuru yigikoresho cya synthesis ntabwo ari ngombwa.Abantu benshi bakora primers mumyaka mirongo kandi ntibazi ko synthesizer ari ABI3900.
Kubijyanye n'amahame yo gushushanya primer, ntugomba kubifata mumutwe ukoresheje rote, kubera ko porogaramu nyinshi zo gushushanya za primer cyangwa ibikoresho byo kumurongo bishobora kwita kuri ibyo bibazo (bisabwa igikoresho cyo kumurongo primer3.ut.ee/), kandi 99,999% byubushakashatsi bwa primer ntibikorwa nintoki Reba, umwanditsi rimwe na rimwe ashushanya amajana yambere kumunsi, niba usomye umwe umwe, bizahinduka ijisho.
Gusa reba ingingo zikurikira nyuma ya primers zateguwe:
1. Shushanya primers hafi ya 3 ′ iherezo: Mugihe cyo gukoresha oligo dT primers ya cDNA ya synthèse ya mbere, urebye uburyo bwo kwandukura inyandiko zinyuranye hamwe nubusugire bwa RNA, primers zabugenewe zigomba gutegurwa hafi ya 3 ′ kugirango zongere imikorere.Koresha ishusho kugirango usobanure gutya (nta buryo bwo kubyumva):

Kuki primers igomba gutegurwa hafi ya 3 ′ iherezo, ntibigomba kuba ukuri
2. Agaciro ka TM: Agaciro ka Tm kari kuri 55-65 ° C (kubera ko ibikorwa bya exonuclease aribyo hejuru kuri 60 ° C), naho GC iri kuri 40% -60%.
3. BLAST: Kugirango wirinde kwongerera umwihariko genome, Blast igomba gukoreshwa mugusuzuma byinyongera.

MIQE (7) —qPCR inzira

1. ibikoresho bya qPCR
Dukurikije ibisabwa na MIQE, tugomba gusobanura neza uko ibintu byifashe byose mu ngingo, harimo iboneza rya sisitemu ya reaction ya PCR, ibikoresho bikoreshwa, ninde wabikoze, uburyo bwa reaction nini, uburyo bwo gusiga irangi cyangwa uburyo bwa probe bwakoreshejwe, igenamigambi rya gahunda ya PCR.Abashoferi b'inararibonye bazabona rwose ko igihe cyose ibikoresho byatoranijwe, amakuru yavuzwe haruguru aragenwa.
Kugeza ubu, gukora no gukora ibicuruzwa bya fluorescent PCR nibikoresho bikuze cyane.Mugihe cyose udahisemo abakora nabi cyane, amahirwe yibibazo ntabwo ari menshi, ariko turashaka gusangira nawe ingingo nke:
Bishyushye-gutangira Taq enzyme:Igice cyingenzi cya PCR nigishyushye-gutangira Taq enzyme.Imisemburo ishyushye-itangira ku isoko muri rusange igabanijwemo ubwoko bubiri, imwe ni enzyme yahinduwe ya chimique itangira (ushobora kubyiyumvisha nka paraffin yashyizwemo), naho ubundi ni enzyme ishyushye yo gutangira guhindura antibody (guhuza antigen-antibody).Guhindura imiti ninzira yambere ya enzymes zishyushye.Iyo ubushyuhe runaka bugeze, enzyme izarekura ibikorwa byayo.Antibody-yahinduwe ashyushye-itangira enzyme ikoresha uburyo bwibinyabuzima kugirango ihagarike ibikorwa bya enzyme.Iyo ubushyuhe runaka bumaze kugerwaho, antibody izahinduka kandi idakora nka poroteyine, kandi ibikorwa bya enzyme bizakinishwa.

Ariko, ubu buryo bukoreshwa iki?Uku niko bimeze, ibikorwa byo kurekura imisemburo ya antibody ihindurwa byihuse kuruta ibya misemburo yahinduwe mu buryo bwa shimi, bityo rero kubijyanye na sensitivite, imisemburo yahinduwe na antibody ifite inyungu nkeya, kuburyo mubusanzwe nta misemburo yahinduwe yimiti mubikoresho ku isoko.Niba ihari, ubwo tekinoroji yuwabikoze iracyatsimbaraye mugihe cyimyaka igihumbi.
Ububiko bwa Magnesium ion:Kwibanda kwa Magnesium ion ni ngombwa cyane mubitekerezo bya PCR.Gukwirakwiza magnesium ion bikwiye birashobora guteza imbere irekurwa ryibikorwa bya Taq enzyme.Niba kwibandaho ari bike cyane, ibikorwa bya enzyme bizagabanuka cyane;niba kwibumbira hamwe ari byinshi, enzyme-catisale idasanzwe idasanzwe izongerwaho.Ubwinshi bwa ioni ya magnesium bizagira ingaruka no kuri primers, ubushyuhe bwo gushonga bwicyitegererezo nibicuruzwa bya PCR, bityo bikagira ingaruka kumusaruro wibice byongerewe.Ubunini bwa ioni ya magnesium bugenzurwa kuri 25mM.Birumvikana, kubikoresho byiza, ubunini bwa ioni ya magnesium bugomba kugenzurwa neza.Abacuruzi bamwe bongeramo magnesium ion chelating agent kuri reagent, ishobora kugera ku ngaruka zo guhinduranya byikora bya magnesium ion.
Irangi rya Fluorescent:Irangi rya Fluorescent, ariryo SYBR Icyatsi dusanzwe dukoresha, cyane cyane ritanga fluorescence muguhuza umwobo muto wa ADN ifite imirongo ibiri, kubera ko guhuza irangi na ADN igizwe n'imirongo ibiri idasanzwe, bivuze ko mugihe cyose ADN ifite imirongo ibiri ihujwe na yo, fluorescence irashobora kubaho, bityo primer-dimers hamwe na ADN byerekana sisitemu bizahuza hamwe na sisitemu.
PS: Bitewe numutungo wacyo woroheje urumuri, ibicuruzwa kumasoko bipakirwa mubituba byijimye bya opaque centrifuge (nkuko bigaragara ku ishusho hepfo).Ariko, ibi bizahura nikibazo.Biragoye kumenya niba amazi yonsa mugihe cyo gutoranya.Ni muri urwo rwego, Qingke mubyukuri yorohereza abakoresha (nkuko bigaragara ku ishusho iri hepfo), kandi umuyoboro ubonerana urapakirwa mu gikapu kitagaragara.Noneho shyira mu gikapu, uzirikana icyoroshye cyo kwirinda urumuri no gutoranya.Ugomba guhitamo umubare wibicuruzwa bikwiye.TSE204 nubuzima buhebuje buhenze, butuma nshaka gutera ibyatsi.

Ubwinshi bwirangi rya fluorescent nabwo ni ngombwa cyane.Niba kwibandaho ari bike cyane, amplification curve ntizamuka murwego rukurikira kandi ntabwo itunganye;niba kwibumbira hamwe ari byinshi, bizatera urusaku.Kubera ko PCR igereranya PCR ahanini ishingiye ku gaciro ka CT, niba kwibumbira hamwe kw'irangi rya fluorescent bidahinduwe neza, ingingo yo hasi iruta iyo hejuru.Birumvikana, kwibanda kumabara akwiye nibyiza.

ROX: Irangi rya ROX rikoreshwa mugukosora neza-neza-neza neza ibimenyetso bya fluorescence.Bamwe mubakora ibikoresho bisaba kalibrasi, mugihe abandi batabikora.Kurugero, gukoresha ibikoresho bya Thermo Fisher Scientific's Real Time PCR yongerera imbaraga mubisanzwe bisaba kalibrasi, harimo 7300, 7500, 7500Fast, StepOnePlus, nibindi. Amabwiriza rusange y'ibikoresho azabisobanura.
QPCR ivanze ya Foregene nayo irimo irangi rya ROX, ryoroshye gukoreshwa muburyo butandukanye.

Igihe nyacyo PCR Kit-Taqman

Intege nke za hydrogène: Kuvura imigozi ya hydrogène idakomeye nikibazo cya tekiniki.Ntakintu cyasomye imfashanyigisho y'ibikoresho byinshi, ariko ntanumwe murimwe wigeze avuga iyi ngingo.Mubyukuri, ni ngombwa cyane.Guhuza shingiro ahanini biterwa nimbaraga za hydrogène.Inkunga ikomeye ya hydrogène ni amplification isanzwe, kandi imigozi ya hydrogène idakomeye iganisha kuri amplification idasanzwe.Niba intege nke za hydrogène zidashobora kuvaho neza, amplification idasanzwe ntishobora kwirindwa.Mubisobanuro byumwanditsi, ibigo bike byabonye iki kibazo.Mugihe uguze ibikoresho, urashobora kwerekeza niba waratekereje kubisubizo muriki kibazo kubikoresho ushaka guhitamo.

Ingano y'ibikorwa: Sisitemu ya 20-50ul ikoreshwa cyane, kandi ingano ntoya ishobora gutera amakosa.Mubisanzwe nukuvuga, amabwiriza yigitabo azagusaba gukoresha ikoreshwa rya PCR.Ntugire ubwenge kandi ukoreshe amajwi mato kugirango uzigame ibiciro.intego ya.Ingano yasabwe nabacuruzi yarageragejwe mubyukuri, kandi birashoboka ko badashobora gukemura ikibazo cyamakosa yatewe numubare muto.
2. Uwayikoze numero yingingo ya plaque
Buriwese azi ihame rya fluorescent quantitative PCR.Ikusanyirizo rya Fluorescence rikorwa cyane cyane binyuze mumutwe wa PCR.Mugihe uhisemo PCR ikoreshwa, witondere ingingo ebyiri: kohereza urumuri rwiza kandi rukwiranye nigikoresho.Mubisanzwe nukuvuga, imbaho ​​nigituba cyibirango byingenzi ni byiza, ariko ugomba guhitamo witonze mubijyanye no guhuza n'imihindagurikire y'ikirere, bitabaye ibyo ntuzashobora gukoresha igikoresho.

4. Ubumenyi bwo murwego rwo hejuru

MIQE (8) —qPCR kwemeza
Nibyingenzi byambere bya qPCR!Intwari nyinshi rero zaguye mumucanga hano.Birumvikana, birashoboka kandi ko ufite amahirwe kandi genes wize ziroroshye, nuko ureremba mu buvumo bwa barafu kumuyaga.Kugenzura amakuru ya qPCR agamije kugerageza kwizerwa ryamakuru.Dutondekanya amakuru akenewe yo kugenzura kuburyo bukurikira:

1.Ikizamini cyihariye
Umwihariko wa intego yo kongera gene igeragezwa mugusuzuma niba ishusho ya electrophoreis ari umurongo umwe;kugenzura;gushonga umurongo kugirango urebe niba ikarita yimpinga ari imwe;enzyme igogorwa ryigenzura nubundi buryo.
Hano, twibanze kuri tasesengura amplification idasanzwe akoresheje uburyo bwo gushonga umurongo.Muri rusange, iyo dushushanya primers, ingano yibicuruzwa bisabwa kuba iri hagati ya 80-200bp, bigatuma ubushyuhe bwo gushonga bwibicuruzwa bya PCR 80-85 ° C.Kubwibyo, niba hari impinga zinyuranye, hagomba kubaho ibindi bicuruzwa bidasanzwe byihariye;niba impinga igaragara munsi ya 80 ° C, mubisanzwe ifatwa nkikigereranyo cya primer;niba impinga igaragara hejuru ya 85 ° C, mubisanzwe ifatwa nkuwanduye ADN cyangwa byinshi bidafite imbaraga zo kongera ibice binini.
Icyitonderwa: Rimwe na rimwe hari impinga imwe gusa kuri 80 ° C.Muri iki gihe, iki gitekerezo kigomba kubahirizwa.Birashoboka ko ibisubizo bya amplification byose ari primer dimers.

Ubusanzwe gushonga umurongo (impinga imwe idafite amplification idasanzwe)

Ikibazo cyo gushonga umurongo uteganijwe (amplificateur idasanzwe ya mpinga zidasanzwe)
Analysis Isesengura ry'imanza】

Hano hari impinga nyamukuru, ariko primer dimer irakomeye
Umurongo umwe wo gushonga umurongo uri ku gishushanyo gikurikira urashobora gushuka byoroshye amaso yawe, ukibwira ko ari igeragezwa ryiza, ariko ibisubizo ni bibi rwose.Muri iki gihe, tugomba kureba ubushyuhe bwo gushonga.Ubushyuhe bwo hejuru buri munsi ya 80 ° C, ni primer-dimer rwose.

Nta gice kigenewe, ibipimo byose bya primer
Hano, murumuna wanjye ntashobora guhagarara.Ifoto iri hepfo nifoto yafashwe na terefone igendanwa noherejwe na scumbag.Reagents yakoresheje nibisanzwe bikoreshwa muruganda.Yahinduye kuva kuri T-prefix imwe ahinduka ikindi T-prefix.Ndatekereza ko umaze kubitekereza.Scumbag yarantakambiye ati: “Reagent yakoreshejwe ku ishusho ya mbere ni nziza cyane, kandi impinga ni imwe.Nyuma, nyuma yo gukoresha reagent wasabye, ihinduka nkishusho ya kabiri, hamwe nimpinga zivanze.Wambabaje.“
Tandukanya ibishushanyo bibiri.Urebye, umwe afite impinga imwe, undi afite impinga ebyiri.Ubuswa, impinga imwe nukuri birumvikana.Nibyo koko?
Ikibi kirenze Dou E, ndamutse nshize amashusho abiri mwishusho hepfo, uzahita wumva.Mubyukuri, twamugaye byoroshye kubwubu bwoko bwishusho.Nyuma yo gusesengura neza, twasanze ko: impinga yumubare wambere iri kuri 75 ° C, ikaba primer dimer rwose;impinga yishusho ya kabiri igaragara kuri 75 ° C na 82 ° C, byibuze hari ibicuruzwa bigaragara.

Amashusho y'ibitekerezo byatanzwe nabanyeshuri
Ikibazo cyibanze rero ntabwo arikibazo cya reagent, ahubwo nikibazo cyo gushushanya primer.Muri icyo gihe, irerekana kandi ko bimwe mubirango binini bidafite ubuziranenge bwicyuma, kandi binagaragaza ibyo murumuna wanjye yavuze mbere: Ntabwo ikirango cya reagent gishyigikira ingingo yawe.Ninyandiko yawe yashyigikiye ikirango cya reagents.Tekereza gusa, niba scumbag idahinduye reagent, amakuru atariyo yoherezwa mukinyamakuru, kandi ibizaba byaba ari amahano.
2. Ct agaciro ko kugenzura ubusa
Ntusobanure, niba igenzura ryuzuye rifite agaciro ka Ct, ntabwo ari umwanda?Ariko, uracyakeneye gusobanukirwa nigenzura ryuzuye rifite agaciro ka Ct.Niba ari NTC, bivuze ko hariho ADN zamahanga nko kwanduza reagent.Niba ari NRT, bivuze ko RNA yakuweho ifite ADN yanduye.
3. Umurongo usanzwe
Harimo imisozi ihanamye no kubara, imikorere ya PCR irashobora kubarwa binyuze muri formula.Ubushakashatsi bwuzuye busaba umusozi wumurongo usanzwe kugirango wegere 3.32, na R² wegera 0.9999.
4. Urwego rufite imbaraga
Ingano yingirakamaro ya reaction ni umurongo.Ukurikije inyandikorugero ikoreshwa mugutanga umurongo usanzwe, urwego rugomba kuba rugizwe byibuze byibura 5 yibitekerezo, kandi ukitondera ihinduka ryindangagaciro za Ct kumurongo mwinshi hamwe na gradients nkeya.
5. Kumenya neza
Impinduka mubisubizo bya qPCR, ni ukuvuga, gusubiramo nabi, ni ukuvuga, kutamenya neza, biterwa nibintu byinshi, harimo ubushyuhe, kwibanda, hamwe nigikorwa.qPCR itomoye muri rusange iba igenzurwa uko umubare wimibare ugabanuka.Byiza cyane mubigeragezo bitandukanijwe, uku guhinduranya tekinike bigomba gutandukana nibinyabuzima bitandukanye, kandi ibinyabuzima byigana birashobora gukemura neza itandukaniro ryibarurishamibare mubisubizo bya qPCR hagati yitsinda cyangwa imiti.By'umwihariko kubisuzumabumenyi, ibyiza-byo-guhuza neza (gusubiramo) kurubuga no kubakoresha bigomba kumenyeshwa.
6. Kumenyekanisha neza na LOD (muri multiplex qPCR)
LOD niyo yibanze cyane kuri 95% byintangarugero nziza zagaragaye.Muyandi magambo, kwibumbira hamwe kwa LOD bikubiye murwego rwo kwigana gene ntigomba kurenga 5% byananiranye.Mugihe ukora analyse ya multiplex qPCR, cyane cyane mugushakisha icyarimwe kumenya ihinduka ryimiterere cyangwa polymorphism, multiplex qPCR ikeneye gutanga ibimenyetso byerekana ko ukuri kwibice byinshi byintego bitabangamiwe numuyoboro umwe, gutahura byinshi hamwe no gutahura imiyoboro imwe hamwe na LOD bigomba kuba bimwe.Cyane cyane iyo genes yibanda cyane hamwe na genes yibanda kuri genes icyarimwe icyarimwe, iki kibazo kigomba kwitabwaho.
Ibibazo nibisubizoMuri rusange, ibibazo bikunze kugaragara muri qPCR byo gukemura byibanda kubintu bikurikira:
· Kwiyongera gukomeye
· Guhitamo bigoye kwibanda kuri primer hamwe nibibazo hamwe na primer-dimers
Ubushyuhe bwa annealing ntabwo ari bwo
· Icyiciro cya kabiri kigira ingaruka kumikorere
amplification idafite akamaro
amplification idasanzwebibaho, muri rusange birasuzumwa niba igishushanyo mbonera kidakwiriye, ariko niba utihutiye guhindura primers, urashobora kubanza kugerageza uburyo bukurikira (ihame naryo rifatanije):
· Kongera ubushyuhe bwa annealing - gerageza gukora intege nke za hydrogen zidashobora gukomeza;
· Gabanya igihe cya annealing no kuramba - gabanya amahirwe ya hydrogène idakomeye;
· Kugabanya kwibanda kuri primer - kugabanya amahirwe yo guhuza primers zirenze urugero n'uturere tutagenewe;
Gukora neza
Ibinyuranyo muburyo budasanzwe bwo kwongerera imbaraga - ubushobozi buke bwo kongera imbaraga, hamwe ningamba zo guhangana nubushobozi buke bwo kongera imbaraga ni ikinyuranyo:
· Kongera igihe cya annealing no kuramba;
· Hindura kuri intambwe eshatu PCR no kugabanya ubushyuhe bwa annealing;
· Kongera ibitekerezo bya primer;
Ps.Kugira ngo ikibazo gikemuke byoroshye, abapfu baracyafite gusoma intangiriro yisosiyete ya reagent kugirango barebe niba hari ikintu gikurura hydrogène idakomeye.
Guhitamo bigoye kwibanda kuri primer hamwe nibibazo hamwe na primer-dimers
Uburyo 1: Muri rusange, amabwiriza ya kit ya qPCR yatanze sisitemu kandi asaba kwibanda kuri primer.
Uburyo 2: Gukemura mugushiraho primer concentration gradient.Ifoto iri hepfo yibwe muri sosiyete kugirango igaragaze.Igishushanyo gikurikira kirerekana ibisubizo byuzuye bya fluorescence byakozwe hamwe na gradients eshatu yibanze (100nM, 250nM, 500nM) hamwe na gradients enye zerekana (0.1ng, 1ng, 10ng, 100ng).Ct agaciro k'ibisubizo byubushakashatsi byateguwe kuburyo bukurikira:

Guhitamo Ibanze Byibanze Guhuza buri primer kwibanda kumurongo ukurikira:

Guhitamo kwibanda kuri primer biragaragara, umubano wumurongo wa primer yibanze ya 100nM na 250nM nibyiza, kandi umubano wumurongo wa primer yibanze ya 500nM urakennye.Muri 100nM na 250nM, Ct agaciro ka 250nM ni ntoya, kubwibyo primer yibanze ni 250nM.Mubisanzwe bikabije primer-dimers irashobora kugaragara mugushonga umurongo.Byagenda bite niba primers zabugenewe zidashobora kwirinda primer-dimers?
Uburyo 3: Mugabanye ingano ya primers kandi wongere ubushyuhe bwa annealing (nta mpamvu yo gusobanura).
Agaciro keza k'ubushyuhe bwa annealing ni 60 ° C.Niba utazi neza, nigute ushobora guhitamo ubushyuhe bukwiye bwa annealing?Igisubizo ni kimwe no guhitamo kwibanda kuri primer -ikizamini cya gradient.Fata ifoto muri sosiyete ya Bio-rad kugirango ugaragaze ikibazo.Kugirango hongerwe igice runaka cyintego, shiraho ubushyuhe bwa gradients umunani, buri hamwe nibisubiramo bitatu, kandi ibyakuweho amplification curve nibi bikurikira:

guhitamo ubushyuhe bwa annealing:
· 70 ° C, 69 ° C - Ahanini, primers ntishobora guhuzwa, ntihabe rero amplification.
· 67.3 ° C - Hano hari umubare muto wa amplification mu ntangiriro, kandi agaciro ka Ct ni nini.
· 64.5 ° C —— Agaciro Ct kagabanuka.
· Kuri 60.7 ° C, 58.0 ° C, 56.2 ° C, na 55.0 ° C, indangagaciro za Ct ahanini wasangaga zihamye, ariko indangagaciro za fluorescence zanyuma zari zitandukanye.
Nigute ushobora guhitamo?Ihame: Ihame rya mbere nigiciro cyo hejuru Ct.Kubintu bimwe bya Ct, hitamo ubushyuhe bwo hejuru bwa annealing kugirango wirinde dimerisation na amplification idasanzwe.Nubwo hari agaciro gakomeye ka fluorescence kuri 55 ° C, hashobora kuba harimo dimers cyangwa amplification idasanzwe muri yo.
Ariko niba uri umunyabwenge nkawe, uzatekereza rwose: Mu buryo bwumvikana, niba reaction ya PCR isobanutse neza, mugihe cyose primer yibanze irenze icyangombwa gisabwa, ingingo ndende kandi ntoya ntigomba kugira ingaruka, nkamabara ya fluorescent na dNTPs.Mubyukuri, mugihe cyose ubushyuhe bwa annealing butezimbere neza, ingaruka yibitekerezo bya primer kuri Ct agaciro bizagabanuka.

Ubushyuhe bwa annealing buteganijwe neza, kandi ingaruka zo kwibanda kuri primer kuri CT zizagabanuka
Icyiciro cya kabiri kigira ingaruka kumikorere
Reka dufate ifoto muri Bio-rad kugirango tugaragaze ikibazo.Irashushanya kandi ubushyuhe bwikigereranyo kugirango yongere gene ifite urwego rwa kabiri.

Imiterere ya kabiri iragaragara
Birashobora kugaragara ko uko ubushyuhe bugenda bugabanuka, ibicuruzwa bitangira kugaragara kandi agaciro ka Ct kagenda imbere, kagera ku giciro gito kuri 60.7 ° C, hanyuma uko ubushyuhe bugenda bugabanuka, agaciro ka Ct kaba nini.Ibinyuranye, uko ubushyuhe bwiyongera, imiterere ya kabiri irakinguka kandi amplification ikora iriyongera.Nyuma yo kugera ku bushyuhe runaka, kongera ubushyuhe ntibishobora kunoza imikorere ya amplification.Kuberako primers idashobora guhuzwa neza muriki gihe.Kubwibyo,reba ubushyuhe hamwe na Ct yo hasi cyane, nubushuhe bwiza bwo kongera inyubako yuburyo bwa kabiri!Nibyo, abapfu bajijutse bagomba kumenya ko niba bidakenewe, nibyiza guhindura primers no kwirinda akarere ka kabiri.
5. Urwego rwo gusaba
MIQE - Isesengura ryamakuru

Isesengura ryamakuru ritangwa cyane cyane nibikoresho bya PCR bya fluorescent.Mu ngingo ibanziriza iyi, imirimo myinshi yo gusesengura amakuru yarakozwe, nko kugenzura ubusa, byasobanuwe mu gishushanyo mbonera.Imbere yerekana gen, gusubiramo imibare, nibindi byasobanuwe., hano turasobanura cyane cyane ikoreshwa rya qPCR.
qPCR ikoreshwa cyane, kandi kugenzura ubushakashatsi hamwe no gusuzuma aside nucleique nibisanzwe bikoreshwa.
Umubare wuzuye
Logika (intangiriro yibanze) ifite umurongo ugereranije numubare wizunguruka.Igipimo gisanzwe gishobora gukurwa mubisanzwe hamwe numubare wambere uzwi wa kopi, ni ukuvuga, umurongo ugereranya umurongo wa amplification reaction urashobora kuboneka.Ukurikije Ct agaciro k'icyitegererezo, kwibanda muri sample birashobora kubarwa.Ingano yinyandikorugero kugirango ushiremo.

Uburyo bwo Kubara Byuzuye
Umubare wuzuye ugomba gushingira kumurongo usanzwe.Gukora umurongo usanzwe, birasabwa.Mubisanzwe, ibisanzwe ni plasmid yabonetse mugukwirakwiza gene intego.Kuki ari plasmid?Kuberako uruziga rwa plasmid ADN arirwo ruhamye.Koresha ibicuruzwa bisanzwe muri gradients 5 kugeza kuri 6 ukurikije igipimo cyikubye kabiri (dilution 10), kandi witondere uburinganire mugihe ucyuye.Reka Ct agaciro kagabanuke hagati ya 15-30.

Gutegura bisanzwe
Muri icyo gihe, icyitegererezo kigomba kugeragezwa nacyo kigomba kuvugururwa bikurikije (ibuka ibintu byoroheje), kandi agaciro ka Ct nako kagomba kugabanuka hagati ya 15-30.Igicuruzwa gisanzwe + icyitegererezo kigeragezwa gishyirwa kumashini hamwe.Nyuma yo kwiruka, umurongo usanzwe wakozwe hamwe nibintu bisanzwe, kandi ibyitegererezo bizageragezwa byazanwe kumurongo usanzwe kugirango ubare intumbero.
Virusi ya Hepatitis B Umubare wa HBV ni umubare wuzuye wuzuye, ushobora kubara umubare wa kopi ya virusi muri 1ml yamaraso.
Kubara nimero ya kopi
Icyitegererezo cyokugeragezwa (ng / ul) = OD260 × 50ug / ml factor ibintu byokugabanuka
Icyitegererezo cy'uburemere = umubare wibanze × 324
Kopi yimibare yicyitegererezo igomba gupimwa (kopi / ul) = ubunini bwikitegererezo bugomba gupimwa / uburemere bwa molekile yicyitegererezo × 6 × 1014

Uburyo bwo kubara nimero ya kopi

Ibyavuzwe haruguru nuburyo bwo kubara kugirango umenye ingano.Iki nikibazo cyimibare gishobora gukemurwa nyuma yo kurangiza amashuri yisumbuye, kandi ibibazo byimibare bikemurwa na mudasobwa.Niba udasobanukiwe, urashobora kuza kuvugana.
Kugereranya
Umubare ugereranije ukoreshwa cyane mubushakashatsi bwa siyansi.Ni virusi zingahe ziri muri 1ml yamaraso, kandi ni virusi ya ADN, iki nikintu gisa nkicyerekana: umubare wamaraso urashobora kumenyekana, kandi virusi ya ADN irahagaze neza.Ariko rero, biragoye kuri twe kugereranya umubare wa kopi ya transcript ya gene runaka mu kibabi, kubera ko bigoye kumenya ingano, uburemere, nubugwaneza bwikibabi, ingano ya RNA yakuweho iragoye kuyimenya, kandi nubushobozi bwo kwandukura inyandiko nayo biragoye kubimenya, nukuvuga ko intambwe iyo ari yo yose ishobora gutuma amakuru yubushakashatsi agira amakosa kandi ntashobora gukoreshwa.
Kubwibyo, kugereranya bigomba kwerekana ikintu:imbere imbere.
Muyandi magambo, kugereranya ugereranije mubyukuri ni igereranya hagati ya gen igenewe na gen imbere.Ugereranije mubice bimwe na selile imwe, ingaruka z'ubunini bw'icyitegererezo, amafaranga yo gukuramo RNA, guhinduranya neza, hamwe na PCR ni bike.Kubera ubunini bw'icyitegererezo, ingirabuzimafatizo zombi hamwe na gen zigenewe byagabanutse.Niyo mpamvu twashimangiye uburinganire n'umutekano mbere.
Imbere yerekana imbere muri rusangeingirabuzimafatizo.
Ntukitiranya iki gitekerezo.Gukora urugo ni amagambo yibinyabuzima, mugihe imbere yimbere ni amagambo ya tekiniki yubushakashatsi.Ingirabuzimafatizo zo mu rugo zigomba gutambuka mbere yo gutoranywa nka gen.
Kurugero, twahisemo ingirabuzimafatizo nyinshi zo mu rugo ku gishushanyo gikurikira kugira ngo dusuzume imiterere yazo mu ngirabuzimafatizo zitandukanye, dusanga urwego rwo kwerekana imiterere ya β-2-microglobuline rutandukanye cyane n’izindi genes eshatu, ku buryo zidashobora gukoreshwa nka gen imbere.

Nyuma yo gusobanukirwa imikorere yo gukosora gene yimbere, algorithm ebyiri zikomoka kuberako kwinjiza gene imbere.
· Uburyo bubiri bwo gutandukanya
· 2 - method Uburyo bwa Ct (uburyo bwo kugereranya agaciro CT)
Niba ushishikajwe no kwiga amoko n'imikorere ya gene, nyamuneka ureke ubushakashatsi kuri algorithm hanyuma ukoreshe formulaire, cyangwa ukoreshe imashini muburyo butaziguye;niba uri umusore ugororotse mu mibare nubuhanga, nyamuneka wumve neza.
uburyo bubiri bwo gutondeka uburyo
Kugereranya intego ya gene hamwe no kubungabunga urugo rwa sample yo kugenzura hamwe nicyitegererezo kigomba kugeragezwa hifashishijwe umurongo usanzwe, hanyuma ubare agaciro kagereranijwe ukurikije formulaire yo kubara, aribwo buryo bwo kugereranya urwego.
Ibyiza: isesengura ryoroshye, ugereranije byoroshye kugerageza
Ibibi: Kuri buri gene, buri cyiciro cyubushakashatsi kigomba gukora umurongo usanzwe
Gushyira mu bikorwa: Bumwe mu buryo bubiri bukoreshwa kandi buzwi ugereranije nuburyo bwo kugereranya muburyo bwo kwiga imvugo ya gene
Inzira niyi ikurikira:

Ingero nizi zikurikira:

Kubara umubare ugereranije ukurikije ibisubizo byuzuye
2 - method Uburyo bwa Ct (uburyo bwo kugereranya agaciro CT)

Ibyiza: Ntabwo ari ngombwa gukora umurongo usanzwe
Ibibi: Bikekwa ko imikorere ya amplification igera hafi 100%;gutandukana bisanzwe ni <5%, kandi umurongo usanzwe hamwe nubushobozi hagati ya buri amplification bifatwa ko bihuye;gutezimbere ibihe byubushakashatsi biragoye.
Gushyira mu bikorwa: Bumwe mu buryo bubiri bukoreshwa kandi buzwi ugereranije nuburyo bwo kugereranya muburyo bwo kwiga imvugo ya gene

Birumvikana ko amplification imikorere isanzwe idashoboka kuba nziza 1. Uburyo bwo gukosora: Niba tuzi ko intego ya gene hamwe na gene yerekanwe bifite ubushobozi bumwe bwo kwongera imbaraga, ariko imikorere ya amplification ntabwo ihwanye na 1, noneho 2－ △△ Ct irashobora gukosorwa nka: (1 + E) - △△ Ct, kurugero, niba imikorere ya amplification ari 0.95, noneho formula yo kubara irashobora gukosorwa kuri 1.95－ △△
Kugeza ubu, ibikubiye kuri fluorescent quantitative PCR byarangiye.