Umwihariko wo gutahura

Mubihe byinshi, intego yo gushushanya primer ni ukugaragaza cyane umwihariko wa PCR.Ibi bigenwa ningaruka nyinshi cyangwa nkeya zishobora guhanurwa zimpinduka nyinshi.Impinduka imwe yingenzi ni urukurikirane kuri 3′end ya primer.

Icy'ingenzi, PCR isobanura igenewe umwihariko birashoboka cyane ko izakomeza gukora neza murwego rwagutse, kubera ko isuzuma ridatanga ibicuruzwa bidasanzwe byongerewe imbaraga, bityo bigahangana na reagent ya PCR cyangwa bikabuza reaction nyamukuru.

Birumvikana ko, mubihe bimwe na bimwe, umwihariko ntabwo aribyingenzi cyane, kurugero, mugihe intego ari ukugereranya isano ya hafi ariko itera virusi zitandukanye, igishushanyo cyihariye, kuzamura no kugenzura birakenewe.

Gushonga umurongo nuburyo busanzwe bwo gusuzuma umwihariko wa amplicons, byibuze ukurikije niba ugomba kongera intego imwe.Ariko, hagomba gushimangirwa ko gushonga umurongo bishobora kuyobya kuko, kurugero, birashobora guterwa ningaruka ziterwa na suboptimal primers hamwe nicyitegererezo gito.

P5 |Gushonga umurongo byerekana Tm ihindagurika yabonetse mubintu bibiri byerekana umubare utandukanye wa ADN ebyiri zigamije.

A. Mugihe cyo hejuru (ad)), nta primer dimer igaragara nyuma yo gupima qPCR irangiye.Nkuko inyandikorugero yibanda igabanuka kuri kopi 50 (e), ibicuruzwa bidasanzwe bitangira kugaragara kandi bihinduka ibicuruzwa byonyine murwego rwo hasi (f).

B. Ikizamini cyanditse Tms imwe kumurongo wose wibanze, kandi nta primer dimer yagaragaye niyo yari yibanze cyane (kopi 5).Iyo ukoresheje ubu buryo bubiri bwo kumenya, nta bicuruzwa byongerewe imbaraga byagaragaye muri NTC.

P5 yerekana imirongo yo gusesa yabonetse hamwe nurugero aho inyandikorugero iba ihari.P 5a yerekana ko kuri bibiri byibuze byibuze, Tms yibicuruzwa bidasanzwe byongerewe umusaruro byakozwe biri munsi yibya amplicons yihariye.

Biragaragara, ubu buryo bwo gutahura ntibushobora gukoreshwa muburyo bwo kumenya intego zihari murwego rwo hasi.

Igishimishije, NTCs, ni ukuvuga, ingero zidafite ADN namba, ntabwo yanditse (ibicuruzwa bidasanzwe) byongera ibicuruzwa, byerekana ko ADN yibinyabuzima ishobora kugira uruhare muburyo budasanzwe bwo kwongera / polymerisiyonike.

Rimwe na rimwe, ama progaramu yibanze hamwe na amplification adasanzwe ntashobora gukosorwa, ariko birashoboka cyane ko dushushanya uburyo bwo gutahura budafite amplification idasanzwe muburyo ubwo aribwo bwose hamwe na NTC (P 5b).

Hano, ndetse no kwandika amplification yintego yibanze hamwe na Cq ya 35 bizatanga umurongo wihariye wo gusesa.Muri ubwo buryo, NTCs nta kimenyetso cyerekana amplification idasanzwe.Rimwe na rimwe, imyitwarire yo gutahura irashobora guterwa n'inzoga za nyina, kandi gusa amplification idasanzwe igaragara mubice bimwe na bimwe bya buffer, bishobora kuba bifitanye isano na Mg2 + zitandukanye.

Guhagarara neza

Gutezimbere Ta ni intambwe yingirakamaro muburyo bwo kugenzura no gutezimbere ibikorwa bya qPCR.Itanga icyerekezo kiziguye cyerekana imbaraga za primer yashyizweho yerekana ubushyuhe (cyangwa ubushyuhe bwubushyuhe) butanga Cq yo hasi cyane itongereye NTC.

Itandukaniro ryibiri kugeza kuri bine muburyo bwo kwiyumvisha ibintu ntibishobora kuba ingenzi kubantu bafite imvugo ndende ya mRNA, ariko kubizamini byo gusuzuma, birashobora gusobanura itandukaniro riri hagati yibisubizo byiza nibinyoma.

Imiterere ya Ta ya qPCR yibanze irashobora gutandukana cyane.Ibizamini bimwe ntabwo bikomeye cyane, kandi nibidakorwa munsi ya Ta nziza nziza ya primers, bizahita bisenyuka.

Ibi ni ngombwa kuko ubu bwoko bwo gutahura bukunze kuba ikibazo kwisi, kandi ubuziranenge bwikitegererezo, kwibumbira hamwe kwa ADN, cyangwa kuba hari izindi ADN ntibishobora kuba byiza.

Mubyongeyeho, intego yo gukoporora umubare irashobora gutandukana muburyo butandukanye, kandi reagent, ibikoresho bya pulasitike, cyangwa ibikoresho bishobora gutandukana nibyakoreshejwe mugihe cyo gushyiraho ikizamini.

P6 | Ubushyuhe buringaniye bwerekana imbaraga zitandukanye zo kumenya PCR.

A. Koresha Sensifast ya Bioline SYBR mastermix (catalog numero BIO-98050) kugirango ukore PCR kuri cDNA yateguwe mubwonko bwabantu RNA.

B. Koresha igikoresho cya CFX qPCR ya Bio-Rad kugirango wandike ikarita ya amplification hamwe no gusesa umurongo wa apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGATCTCCTAGG).

C. Igishushanyo mbonera no gushonga umurongo wa ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).

D. Igishushanyo mbonera no gusenya umurongo wa GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGATCTTC).

E. Cqs yanditse ku bushyuhe butandukanye bwa annealing, yerekana itandukaniro muri Cq ryanditswe munsi yubushyuhe bwa 7C.

P 6 yerekana ibisubizo bisanzwe byikizamini kitifuzwa, aho qPCR yakorewe ikoresheje Tasdi ya Tasdi iri hagati ya 59C na 67C (P 6a), ikoresheje primers kuri genes eshatu zihariye zubwonko bwabantu.

Birashobora kugaragara mubishushanyo mbonera byerekana ko Opalin primers iri kure yicyiza kuko urutonde rwiza rwa Ta ni ruto cyane (Igishusho 6b), ni ukuvuga ko Cqs yatatanye cyane, bigatuma Cqs igereranywa cyane na Cqs nziza.

Ubu buryo bwo gutahura ntabwo buhagaze kandi bushobora kuganisha kuri suboptimal amplification.Kubwibyo, iyi couple ya primers igomba gusubirwamo.Mubyongeyeho, gusesengura umurongo wo gusesengura (inset) byerekana ko umwihariko wubu buryo bwo gutahura ushobora no kuba ikibazo, kuko gushonga umurongo wa buri Ta uratandukanye.

Uburyo bwo kumenya ACSBG1 bwerekanwe muri P 6c burakomeye kuruta uburyo bwa Opalin bwo gutahura hejuru, ariko buracyari kure yicyiza, kandi birashoboka ko bushobora kunozwa.

Ariko, dushimangira ko nta sano rikenewe riri hagati yubukomezi nuburyo bwihariye, kubera ko umurongo wo gusesa wakozwe nubu buryo bwo gutahura werekana agaciro kamwe kamwe muri Tas (inset).

Kurundi ruhande, ikizamini gikomeye kirashobora kwihanganira cyane, gitanga Cqs isa murwego runini rwa Tas, nko mubizamini bya GFAP byerekanwe muri P 6d.

Itandukaniro muri Cqs ryabonetse muri dogere 8 selisiyusi imwe iri munsi ya 1, kandi umurongo wo gusesa (inset) wemeza ibimenyetso biranga muri ubu bushyuhe.Birakwiye ko tumenya ko Tas yabazwe hamwe na Ta nyayo ishobora kuba itandukanye cyane.

Hariho amabwiriza menshi yagenewe gufasha abashakashatsi gukora primers neza, inyinshi murizo zishingiye kumategeko amaze igihe kirekire kandi hitabwa cyane kuri 3′end ya primers.Birasabwa kenshi gushyiramo G cyangwa C kuri 3 'iherezo na bibiri bya G cyangwa C (clamp ya GC), ariko ntibirenze bibiri mubice 5 byanyuma.

Mubikorwa, aya mategeko arashobora kuyobora abashakashatsi, ariko ntabwo byanze bikunze arikosora mubihe byose.

P7 |3′end ya primer ntigira ingaruka nke muburyo bwihariye cyangwa gukora neza.

A. Umwanya wa primers kuri muntu HIF-1α (NM_181054.2).

B. Koresha Agilent Brilliant III SYBR Inzoga zicyatsi kibisi (Cat. No 600882) kugirango wongere ibintu bitandatu byipimishije.

C. Igishushanyo mbonera no gushonga umurongo byanditswe na Bio-Rad's CFX qPCR igikoresho na 3′end primers.NTCs zerekanwa mumutuku.

D. Cqs inyandiko ya buri kintu cyizamini

Kurugero, ibisubizo muri P 7 bivuguruza itegeko rya 3′end.Ibishushanyo byose bitanga ahanini ibisubizo bimwe, hamwe na primer ebyiri gusa ziganisha kuri amplification idasanzwe muri NTC.

Ariko, ntidushobora gushyigikira ingaruka za clip ya GC, kuko muriki gihe, gukoresha A cyangwa T nkibintu 30 ntarengwa ntabwo bigabanya umwihariko.

Ikizamini C, aho F primer irangirira muri GGCC, yanditse Cqs muri NTCs, byerekana ko umuntu ashobora gushaka kwirinda izo gahunda kuri 30-mpera.Turashimangira ko inzira yonyine yo kumenya ibyiza 3′end bikurikirana bya primer couple ni ugusuzuma abakandida primers mubigeragezo.

Gukora neza

Icy'ingenzi, nubwo PCR idasobanutse neza idashobora na rimwe kuba umwihariko, imikorere ya amplification irashobora guhinduka kandi ikagurwa muburyo bwinshi butandukanye uhindura enzyme, inzoga za nyina, inyongeramusaruro, hamwe nuburyo bwo gusiganwa ku magare.

Kugirango usuzume imikorere ya PCR yo kumenya, nibyiza gukoresha ikwirakwizwa ryikubye inshuro 10 cyangwa 5 acide nucleic aside, ni ukuvuga "uburyo busanzwe bwo gutondeka".

Niba PCR amplicons cyangwa intego ya ADN ya syntetique ikoreshwa mugutanga umurongo usanzwe, ihindagurika ryuruhererekane rwizo ntego rigomba kuvangwa nubunini buhoraho bwa ADN (nka ADN genomic).

P8 |Gukata umurongo kugirango dusuzume imikorere ya PCR.

Igisubizo.

B. 100 ng RNA yahinduwe yandukuwe, yongerwaho inshuro 2, hanyuma urugero rwa cDNA rwakuweho inshuro 5 kugeza kuri 1 ng ADN ya genomic muntu.Gushonga umurongo byerekanwe muri inset.

C. Imyitwarire ya RT, kuyungurura, no gukurikiranwa byasubiwemo icyitegererezo cya kabiri cDNA, kandi ibisubizo byari bisa.

P 8 yerekana imirongo ibiri isanzwe, ukoresheje uburyo bumwe bwo gutahura kuri sample ebyiri zitandukanye za cDNA, ibisubizo nuburyo bumwe, hafi 100%, kandi agaciro ka R2 nako karasa, ni ukuvuga urwego rwo guhuza amakuru yubushakashatsi n'umurongo wo gusubira inyuma cyangwa amakuru Impamyabumenyi yumurongo.

Imirongo ibiri isanzwe iragereranywa, ariko ntabwo ari kimwe.Niba ikigamijwe ari ukugereranya neza intego, hagomba kumenyekana ko bitemewe gutanga umubare wimibare utabanje gusobanura ukutamenya neza

P9 |Ibipimo bidashidikanywaho bijyana no kugereranya ukoresheje umurongo usanzwe.

A. Koresha primers kuri GAPDH (NM_002046) kugirango ukore PCR no gushonga umurongo.F.

B. Imbonerahamwe yerekana imbaraga, gushonga umurongo hamwe nu murongo usanzwe wanditswe hamwe na Bio-Rad ya CFX qPCR igikoresho.

C. Igishushanyo mbonera gisanzwe hamwe na 95% intera yicyizere (CI).

D. Kopi nimero na 95% intera intera ya Cq eshatu zikomoka kumurongo wo kugabanuka.

P 9 yerekana ko kubizamini byateguwe neza, ihinduka ryihariye ryumurongo umwe usanzwe ni inshuro zigera kuri 2 (95% intera yicyizere, byibuze kugeza kuri ntarengwa), bishobora kuba impinduka ntoya ishobora gutegurwa.

Ibicuruzwa bifitanye isano:

Akagari kayobora RT qPCR Kit

Imbeba umurizo Direct PCR kit

Inyama zinyamanswa Direct PCR kit