RT-qPCR yatejwe imbere nubuhanga busanzwe bwa PCR.Yongera imiti ya fluorescent (amarangi ya fluorescent cyangwa fluorescent probe) muri sisitemu gakondo ya PCR, kandi ikamenya inzira ya PCR yo kwagura no kwagura mugihe nyacyo ukurikije uburyo bwabo butandukanye bwa luminescent.Ibimenyetso bya Fluorescent bihinduka muburyo bukoreshwa mukubara ingano yibicuruzwa muri buri cyiciro cya PCR.Kugeza ubu, uburyo bukunze kugaragara ni uburyo bwo gusiga irangi rya fluorescent hamwe nuburyo bwa probe.

Uburyo bwo gusiga irangi rya Fluorescent:
Amabara amwe ya fluorescent, nka SYBR Icyatsi Ⅰ, PicoGreen, BEBO, nibindi, ntabwo bitanga urumuri wenyine, ahubwo bisohora fluorescence nyuma yo guhambira kumurongo muto wa dsDNA.Kubwibyo, mugitangira reaction ya PCR, imashini ntishobora kumenya ibimenyetso bya fluorescent.Iyo reaction igeze kuri annealing-kwaguka (uburyo bwintambwe ebyiri) cyangwa kwaguka (uburyo bwintambwe eshatu), imirongo ibiri irakingurwa muriki gihe, hamwe na polymerase nshya ya ADN Mugihe cyo guhuza imirongo, molekile ya fluorescent ihujwe na dsDNA ntoya hanyuma ikarekura fluorescence.Mugihe umubare wizunguruka PCR wiyongera, amarangi menshi kandi menshi ahuza na dsDNA, kandi ibimenyetso bya fluorescent nabyo bikomeza kwiyongera.Fata SYBR Icyatsi Ⅰ nkurugero.
Uburyo bw'ubushakashatsi:
Taqman probe niyo hydrolysis ikoreshwa cyane.Hano hari itsinda rya fluorescent kuri 5 ′ iherezo ryiperereza, mubisanzwe FAM.Iperereza ubwaryo ni urukurikirane rwuzuzanya na gen.Hano hari itsinda ryazimya fluorescent kuri 3 ′ iherezo rya fluorophore.Ukurikije ihame ryo guhererekanya ingufu za fluorescence (kohereza ingufu za Förster resonance, FRET), mugihe itsinda ryumunyamakuru fluorescent (molekile ya donor fluorescent) hamwe nitsinda rya florescent rizimya (molekile ya fluorescent) Iyo umunezero ushimishije urenze kandi intera iri hafi cyane (7-10nm), umunezero wa molekile yabaterankunga urashobora gutera fluorescence ya molekile ya nyirubwite.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.

Igishushanyo 1 Ihame rya qRT-PCR

Igishushanyo mbonera
Amahame:

Primers igomba kuba yarateguwe mukarere kabitswe ka acide nucleic kandi ifite umwihariko.

Nibyiza gukoresha cDNA ikurikirana, kandi mRNA ikurikirana nayo iremewe.Niba atari byo, shakisha igishushanyo cya cds akarere ka ADN ikurikirana.
Uburebure bwibicuruzwa bya fluorescent ni 80-150bp, birebire ni 300bp, uburebure bwa primer muri rusange buri hagati ya 17-25, kandi itandukaniro riri hagati yimbere na epfo na ruguru ntigomba kuba nini cyane.

Ibirimo G + C biri hagati ya 40% na 60%, naho 45-55% nibyiza.
Agaciro TM kari hagati ya dogere 58-62.
Gerageza wirinde primer dimers na self-dimers, (ntugaragare kurenza ibice bibiri byikurikiranya byuzuzanya) imiterere yimisatsi, niba bidashoboka, kora ΔG <4.5kJ / mol * Niba udashobora kwemeza ko gDNA yakuweho mugihe cyo kwandukura bisukuye, nibyiza gushushanya primers ya intron * 3 ′ iherezo ntishobora guhindurwa, kandi wirinde G / GC,
Umwihariko Homologiya ya heterogeneously amplified series ikurikiranye neza ni munsi ya 70% cyangwa ifite 8 byuzuzanya shingiro.
Ububikoshingiro:
Gushakisha PambaFGD ukoresheje ijambo ryibanze
Igishushanyo mbonera:
Igishushanyo mbonera cya IDT-qPCR

Igishushanyo2 IDT kumurongo wigikoresho cya primer

Ishusho3 ibisubizo byerekana urupapuro
Igishushanyo mbonera cya lncRNA:
lncRNA:intambwe imwe na mRNA.
miRNA:Ihame ryuburyo bwa stem-loop: Kubera ko miRNAs zose ari urukurikirane rugufi rwa 23 nt, gutahura PCR itaziguye ntishobora gukorwa, bityo igikoresho gikurikirana stem-loop gikoreshwa.Urutonde rwa stem-loop ni ADN imwe igizwe na 50 nt, ishobora gukora imisatsi yonyine.3 'Iherezo rishobora gushushanywa nkurukurikirane rwuzuzanya nigice cya miRNA igice, hanyuma intego miRNA irashobora guhuzwa nurwego rwa stem-loop mugihe cyo kwandukura, kandi uburebure bwose bushobora kugera kuri 70bp, bujyanye nuburebure bwibicuruzwa byongerewe byagenwe na qPCR.Kudoda miRNA igishushanyo mbonera.
Amplification-yihariye gutahura:
Ububiko bwo kumurongo kuri interineti: CottonFGD iturika ukurikije urutonde
Igisasu cyaho: Reba gukoresha Blast + kugirango ukore ibisasu byaho, linux na macos birashobora gushiraho byimazeyo base base, sisitemu ya win10 nayo irashobora gukorwa nyuma yo gushiraho ubuntu bash.Kora base base base hamwe nibisasu byaho;fungura ubuntu bash kuri win10.
Icyitonderwa: Ipamba yo hejuru hamwe nizinga ryinyanja ni ibihingwa bya tetraploid, kubwibyo ibisubizo byo guturika bizaba inshuro ebyiri cyangwa nyinshi.Mubihe byashize, gukoresha cd ya NAU nkububiko kugirango ukore ibisasu birashoboka ko uzabona genes ebyiri za homologique zifite itandukaniro rya SNP gusa.Mubisanzwe, genes ebyiri zombi ntizishobora gutandukanywa nigishushanyo mbonera, bityo zifatwa nkimwe.Niba hari indel igaragara, primer isanzwe ikorerwa kuri indel, ariko ibi birashobora kuganisha kumiterere ya kabiri ya primer Ingufu zubusa ziba nyinshi, bigatuma kugabanuka kwingirakamaro, ariko ibi ntibishoboka.

Kumenya primer yuburyo bwa kabiri:
Intambwe:fungura oligo 7 → kwinjiza inyandikorugero zikurikirana → gufunga idirishya → kuzigama → gushakisha primer ku gishushanyo, kanda ctrl + D kugirango ushireho uburebure bwa primer → gusesengura ibice bitandukanye byisumbuyeho, nko kwiyobora umubiri, heterodimer, umusatsi, kudahuza, nibindi. Amashusho abiri yanyuma mubishusho bya primers.Igisubizo cya primer y'imbere ni cyiza, nta miterere igaragara ya dimer na hairpin igaragara, nta shingiro ryuzuzanya rihoraho, kandi agaciro ntarengwa k'ingufu z'ubuntu kari munsi ya 4.5, mugihe primer yinyuma yerekana ikomeza Ibishingiro 6 byuzuzanya, kandi ingufu z'ubuntu ni 8.8;mubyongeyeho, dimer irenze igaragara kuri 3 ′ iherezo, naho dimer ya 4 ikurikiranye igaragara.Nubwo ingufu zubuntu zitari hejuru, 3 ′ dimer Chl irashobora kugira ingaruka zikomeye kumikorere no kongera imbaraga.Mubyongeyeho, birakenewe kugenzura imisatsi, heterodimers, hamwe nibidahuye.

Igishushanyo cya oligo7 ibisubizo
Kumenyekanisha imikorere neza:
Kwiyongera kwa reaction ya PCR bigira ingaruka zikomeye kubisubizo bya PCR.Muri qRT-PCR, imikorere ya amplification ningirakamaro cyane kubisubizo byuzuye.Kuraho ibindi bintu, imashini na protocole muri reaction ya buffer.Ubwiza bwa primers nabwo bugira uruhare runini kumikorere ya amplification ya qRT-PCR.Kugirango hamenyekane neza ibisubizo byukuri, ingano ya fluorescence igereranijwe hamwe numubare wuzuye wa fluorescence ukeneye kumenya imikorere ya amplification ikora neza.Birazwi ko QRT-PCR ikora neza iri hagati ya 85% na 115%.Hariho uburyo bubiri:
1. Uburyo busanzwe bwo guta umurongo:
a.Kuvanga cDNA
b.Kugabanuka gahoro gahoro
c.qPCR
d.Ikigereranyo cyo gusubira inyuma kugereranya kubara neza
2. LinRegPCR
LinRegPCR ni porogaramu yo gusesengura igihe nyacyo RT-PCR Data, nanone yitwa data PCR (qPCR) ishingiye kuri SYBR Green cyangwa chimie isa nayo.Porogaramu ikoresha amakuru adashingiye ku makuru yakosowe, ikora ubugororangingo bwibanze kuri buri cyitegererezo Bitandukanye, igena idirishya-ryumurongo hanyuma ikoresha isesengura ryisubiramo kugirango rihuze umurongo ugororotse unyuze muri data ya PCR.Uhereye kumurongo wuyu murongo PCR ikora neza ya buri cyitegererezo cyabazwe.Hagati ya PCR ikora neza kuri amplicon na Ct agaciro kuri sample ikoreshwa mukubara intangiriro yo gutangira kuri sample, bigaragarira mubice bya fluorescence uko bishakiye.Ibyinjijwe nibisohoka binyuze muri Excel urupapuro.Icyitegererezo gusa
kuvanga birakenewe, nta gradient
intambwe zirakenewe:(Fata Bole CFX96 nkurugero, ntabwo Imashini rwose ifite ABI isobanutse)
igerageza:ni igeragezwa risanzwe rya qPCR.
QPCR yasohotse:LinRegPCR irashobora kumenya uburyo bubiri bwibisohoka dosiye: RDML cyangwa kugereranya Amplification ibisubizo.Mubyukuri, nigiciro nyacyo cyo kumenya umubare wumuzenguruko na signal ya fluorescence ukoresheje imashini, kandi amplification iboneka mugusesengura agaciro ka fluorescence ihinduka ryumurongo ugizwe neza.
Guhitamo amakuru: Muri théorie, agaciro ka RDML kagomba gukoreshwa.Bigereranijwe ko ikibazo cya mudasobwa yanjye ari uko software idashobora kumenya RDML, bityo mfite agaciro keza cyane nkamakuru yambere.Birasabwa gukora igenzura ryibanze ryamakuru mbere, nko kunanirwa kongeramo ingero, nibindi. Ingingo zishobora gusibwa mumibare yasohotse (birumvikana ko udashobora kuzisiba, LinRegPCR izirengagiza izi ngingo mubyiciro bizakurikiraho)

Igicapo 5 qPCR yohereza hanze

Igishushanyo cya 6 cyo gutoranya abakandida

Ibyinjijwe:Fungura impamyabumenyi yujuje ibyangombwa.xls, → fungura LinRegPCR → dosiye → soma uhereye kuri excel → hitamo ibipimo nkuko bigaragara ku gishushanyo 7 → OK → kanda umenye ibyingenzi

Igishushanyo cya 7 intambwe ya linRegPCR yinjiza amakuru

Igisubizo:Niba nta gusubiramo, nta matsinda asabwa.Niba haribisubirwamo, itsinda rishobora guhindurwa muburyo bw'icyitegererezo, kandi izina rya gene ryinjiye mubiranga, hanyuma gene imwe izahita ishyirwa hamwe.Hanyuma, kanda kuri dosiye, wohereze hanze, hanyuma urebe ibisubizo.Kwiyongera kwa amplification hamwe na R2 ibisubizo bya buri riba bizerekanwa.Icyakabiri, niba ugabanije mumatsinda, impuzandengo ikosowe igereranijwe izerekanwa.Menya neza ko amplification imikorere ya buri primer iri hagati ya 85% na 115%.Niba ari binini cyane cyangwa bito cyane, bivuze ko imikorere ya amplification ya primer ari mibi.

Igishushanyo 8 Ibisubizo nibisohoka

Inzira y'ubushakashatsi:
Ibisabwa bya RNA:
Isuku:1.72.0 yerekana ko hashobora kubaho isothiocyanate isigaye.Acide nucleic aside isukuye A260 / A230 igomba kuba hafi 2 .Niba hariho kwinjiza cyane kuri 230 nm, byerekana ko hariho ibinyabuzima kama nka ion ya fenate.Mubyongeyeho, irashobora gutahurwa na 1.5% agarose gel electrophorei.Erekana akamenyetso, kubera ko ssRNA idafite gutandukana kandi uburemere bwa molekuline logarithm ntabwo ifitanye isano, kandi uburemere bwa molekile ntibushobora kugaragazwa neza.Kwibanda: Mubyukurintabwomunsi ya 100ng / ul, niba kwibumbira hamwe ari bike, ubuziranenge muri rusange ntabwo buri hejuru

Igishusho9 RNA gel

Mubyongeyeho, niba icyitegererezo gifite agaciro kandi RNA ikaba ari ndende, birasabwa kuyigabanya nyuma yo kuyikuramo, hanyuma ukayungurura RNA kugeza kuri 100-300ng / ul kugirango yandukure.Muriinzira yo kwandukura, iyo mRNA yandukuwe, oligo (dt) primers ishobora guhuza byumwihariko umurizo wa polyA ikoreshwa muguhindura transcription, mugihe lncRNA na circRNA ikoresha primers ya hexamer (Random 6 mer) primers kugirango ihindure byimazeyo RNA yose Kuri miRNA, miRNA yihariye ijosi-loop primers ikoreshwa muburyo bwo kwandukura.Ubu ibigo byinshi byatangije ibikoresho byihariye byo kudoda.Kuburyo bwa stem-loop, uburyo bwo kudoda buroroshye cyane, bwinjiza cyane, kandi bukiza-reagent, ariko Ingaruka zo gutandukanya miRNAs yumuryango umwe ntizigomba kuba nziza nkuburyo bwa stem-loop.Buri cyuma gisubiramo ibikoresho bisabwa kugirango umuntu yibanze kuri gene yihariye (stem-loop).Imbere ikoreshwa kuri miRNA ni U6.Muburyo bwo guhinduranya stem-loop, umuyoboro wa U6 ugomba guhindurwa ukundi, naho imbere ninyuma ya U6 bigomba kongerwaho muburyo butaziguye.Byombi circRNA na lncRNA birashobora gukoresha HKGs nkibisobanuro byimbere.MuricDNA gutahura,
niba ntakibazo na RNA, cDNA nayo igomba kuba nziza.Ariko, niba gukurikiranwa gutunganijwe neza, nibyiza gukoresha gene yimbere (Reference gene, RG) ishobora gutandukanya gDNA na cds.Mubisanzwe, RG ni gene yo murugo., HKG) nkuko bigaragara ku gishushanyo cya 10;Muri kiriya gihe, nakoraga proteine ​​yo kubika soya, kandi nkoresha actin7 irimo intore nkimbere.Ingano yagace kongerewe igice cyiyi primer muri gDNA yari 452bp, kandi niba cDNA yarakoreshejwe nkicyitegererezo, yari 142bp.Hanyuma ibisubizo byikizamini byagaragaye ko Igice cya cDNA cyanduye koko na gDNA, kandi cyanagaragaje ko ntakibazo cyatewe nigisubizo cyo kwandukura, kandi gishobora gukoreshwa nkicyitegererezo cya PCR.Ntabwo bimaze gukoresha agarose gel electrophorei hamwe na cDNA, kandi ni bande ikwirakwizwa, ntabwo yemeza.

Igishushanyo cya 10 cDNA

Kugena imiterere ya qPCRmuri rusange ntakibazo ukurikije protocole ya kit, cyane cyane murwego rwa tm agaciro.Niba primers zimwe zidakozwe neza mugihe cyo gushushanya primer, bikavamo itandukaniro rinini hagati yagaciro ka tm na theoretical 60 ° C, birasabwa ko cDNA Nyuma yintangarugero zivanze, koresha progaramu ya PCR hamwe na primers, hanyuma ugerageze kwirinda gushyiraho ubushyuhe butagira imirongo nkigiciro cya TM.

Isesengura ryamakuru

Ubusanzwe isano ya fluorescence igereranya uburyo bwo gutunganya PCR muburyo bwa 2-ΔΔCT.Inyandikorugero yo gutunganya amakuru.

Ibicuruzwa bifitanye isano:

Igihe nyacyo PCR Biroroshye^TM –Taqman

Igihe nyacyo PCR Biroroshye^TM –SYBR GREEN I.

RT Byoroshye I (Master Premix kumurongo wambere cDNA synthesis)

RT Byoroshye II (Master Premix kumurongo wambere cDNA synthesis ya qPCR)