1. Ubumenyi bwibanze (niba ushaka kubona igice cyubushakashatsi, nyamuneka wimure igice cya kabiri)
Nkibisubizo bikomoka kuri PCR isanzwe, Igihe nyacyo PCR ikurikirana cyane cyane ihinduka ryibicuruzwa byongerewe imbaraga muri buri cyiciro cyibisubizo bya PCR mugihe nyacyo binyuze muguhindura ibimenyetso bya fluorescence, kandi ikanasesengura umubare wintangarugero itangira binyuze mumibanire hagati ya ct nagaciro gasanzwe.
Amakuru yihariye ya RT-PCR nishingiro, urubuga rwa fluorescencenaCt agaciro.
| shingiro: | Agaciro ka fluorescence yumuzingi wa 3-15 niwo shingiro (baseline), biterwa nikosa rimwe na rimwe ryo gupimwa. |
| Imipaka (imbago): | Yerekeza kumupaka wa fluorescence yashyizweho kumwanya ukwiye mukarere gakura kerekana akarere ka amplification curve, mubisanzwe inshuro 10 gutandukana bisanzwe. |
| CT agaciro: | Numubare wizunguruko wa PCR mugihe agaciro ka fluorescence muri buri reaction ya reaction igera kumurongo. Agaciro Ct gahwanye nubunini bwintangarugero. |
Uburyo busanzwe bwo kuranga kuri RT-PCR:
| buryo | akarusho | kubura | ingano ya porogaramu |
| SYBR GreenⅠ | Byagutse cyane, byoroshye, bihendutse kandi byoroshye | Ibisabwa byibanze ni byinshi, bikunda kugaragara bidasanzwe | Irakwiriye gusesengura ingano ya genes zitandukanye, ubushakashatsi ku mvugo ya gene, n'ubushakashatsi ku nyamaswa n'ibimera bya transgenji. |
| TaqMan | Umwihariko mwiza no gusubiramo cyane | Igiciro ni kinini kandi gikwiranye nintego zihariye. | Gutahura indwara ya virusi, ubushakashatsi bwokwirinda ibiyobyabwenge, gusuzuma ibiyobyabwenge, gusuzuma indwara zikomoka. |
| urumuri rwa molekile | Umwihariko, fluorescence, inyuma yinyuma | Igiciro ni kinini, birakwiriye gusa kubwintego yihariye, igishushanyo kiragoye, kandi igiciro kiri hejuru. | Isesengura ryihariye rya gen, isesengura rya SNP |
2. Intambwe zigeragezwa
2.1 Ibyerekeye amatsinda yo kugerageza- hagomba kuba amariba menshi mumatsinda, kandi hagomba kubaho gusubiramo biologiya.
| ① | Kugenzura neza | Byakoreshejwe kugirango tumenye imikurire yimikorere mubushakashatsi |
| ② | Kugenzura nabi siRNA (urutonde rwa siRNA idasanzwe) | Erekana umwihariko wibikorwa bya RNAi.siRNA irashobora gutera igisubizo kidasanzwe kubibazo bya 200nM. |
| ③ | Igenzura ryimikorere | Kuramo uburozi bwo kwanduza reagent kuri selile cyangwa ingaruka ku mvugo ya gen igamije |
| ④ | siRNA kurwanya gen | Kuraho imvugo ya gen igenewe |
| ⑤ (bidashoboka) | siRNA | Byakoreshejwe mugukemura sisitemu yubushakashatsi nibibazo byimikorere |
| ⑥ (bidashoboka) | Igenzura rya florescent siRNA | Imikorere yo kwanduza selile irashobora kugaragara hamwe na microscope |
2.2 Amahame yo gushushanya primer
| Ingano y'ibice byongerewe | Byaba byiza kuri 100-150bp |
| Uburebure bwa Primer | 18-25bp |
| Ibirimo GC | 30% -70%, nibyiza 45% -55% |
| Tm agaciro | 58-60 ℃ |
| Urukurikirane | Irinde T / C gukomeza;A / G bikomeje |
| Urutonde rwanyuma | Irinde abakire GC cyangwa AT abakire;iherezo rya base ni byiza G cyangwa C;nibyiza kwirinda T. |
| Kwuzuzanya | Irinde kuzuzanya gukurikiranye kurenze 3 shingiro muri primer cyangwa hagati ya primers ebyiri |
| Umwihariko | Koresha ibisasu kugirango wemeze primer yihariye |
①SiRNA ni ubwoko bwihariye, kandi uko amoko akurikirana azaba atandukanye.
②SiRNA ipakiwe mu ifu yumye, ishobora kubikwa neza ibyumweru 2-4 mubushyuhe bwicyumba.
2.3 Ibikoresho cyangwa reagent bigomba gutegurwa hakiri kare
| Primer (imbere imbere) | Harimo imbere no guhindura bibiri |
| Primers (intego ya gene) | Harimo imbere no guhindura bibiri |
| Intego Si RNA (imirongo 3) | Mubisanzwe, isosiyete izahuza imirongo 3, hanyuma ihitemo imwe muri eshatu na RT-PCR |
| Igikoresho cyo kwimura | Lipo2000 nibindi |
| RNA Ibikoresho Byihuta | Kubikuramo RNA nyuma yo kwimurwa |
| Igikoresho cyihuta cyo Kwandukura | ya synthesis ya cDNA |
| PCR Amplification Kit | 2 × Super SYBR Icyatsi QPCR Umwigisha |
2.4 Kubijyanye nibibazo bigomba kwitabwaho muburyo bwihariye bwo kugerageza:
①siRNA inzira yo kwanduza
1. Kubisahani, urashobora guhitamo isahani 24-isahani, isahani 12-isahani 6-isahani (impuzandengo ya RNA yibanze muri buri riba ryisahani 24 ni 100-300 ng / uL), kandi ubwinshi bwokwirakwiza kwingirabuzimafatizo bugera kuri 60% -80% cyangwa birenga
2. Intambwe zo kwimura nibisabwa byihariye bikurikiza amabwiriza.
3. Nyuma yo kwandura, ingero zirashobora gukusanywa mugihe cyamasaha 24-72 kugirango tumenye mRNA (RT-PCR) cyangwa kumenya proteine mumasaha 48-96 (WB)
Process Uburyo bwo gukuramo RNA
1. Irinde kwanduzwa na enzymes zidasanzwe.Harimo cyane cyane kwambara masike na gants;ukoresheje inama ya sterile sterile na EP tubes;amazi yakoreshejwe mubigeragezo agomba kuba RNase-Yubusa.
2. Birasabwa gukora inshuro ebyiri nkuko byasabwe mubikoresho byo gukuramo byihuse, bizamura rwose ubuziranenge numusaruro.
3. Amazi yimyanda ntagomba gukora ku nkingi ya RNA.
Quant Umubare wa RNA
RNA imaze gukurwa, irashobora kubarwa neza na Nanodrop, kandi gusoma byibuze birashobora kuba munsi ya 10ng / ul.
④Gusubiramo inzira yo kwandukura
1. Bitewe nubukangurambaga bukabije bwa RT-qPCR, hagomba gukorwa byibura amariba 3 abangikanye kuri buri cyitegererezo kugirango Ct ikurikiraho itandukana cyane cyangwa SD kugirango ibe nini cyane kubisesengura mibare.
2. Ntugahagarike kandi ushongeshe Master kuvanga inshuro nyinshi.
3. Buri muyoboro / umwobo ugomba gusimburwa ninama nshya!Ntukomeze gukoresha inama imwe ya pipette kugirango wongere ingero!
4. Filime yometse ku isahani 96-nziza nyuma yo kongeramo icyitegererezo igomba koroherezwa nisahani.Nibyiza gushira centrifuge mbere yo kuyishyira kumashini, kugirango amazi yo kurukuta rwa tube ashobore kumanuka no gukuraho umwuka mubi.
AnalysisGusesengura bisanzwe
| Nta gihe cyo gukura kwa logarithmic | Birashoboka cyane kwibanda kumurongo |
| Nta gaciro ka CT | Intambwe zitari zo zo kumenya ibimenyetso bya fluorescent; gutesha agaciro primers cyangwa probe - ubunyangamugayo bwayo burashobora kugaragara na PAGE electrophoreis; umubare udahagije w'icyitegererezo; gutesha agaciro inyandikorugero - kwirinda kwinjiza umwanda no gukonjesha inshuro nyinshi no gukonjesha mugutegura icyitegererezo; |
| Ct> 38 | Gukoresha imbaraga nke;Ibicuruzwa bya PCR ni birebire cyane;ibice bitandukanye bya reaction byangiritse |
| Umurongo wo kwongera umurongo | Ibibazo birashobora guteshwa agaciro kubice byinshi bikonje cyangwa bikabije kumurika |
| Itandukaniro mubyobo byigana ni binini cyane | Igisubizo cyibisubizo ntabwo gishonga rwose cyangwa igisubizo cyibisubizo ntabwo kivanze;kwiyuhagira ubushyuhe bwigikoresho cya PCR byandujwe nibintu bya fluorescent |
2.5 Ibyerekeye isesengura ryamakuru
Isesengura ryamakuru ya qPCR rishobora kugabanywa mukigereranyo ugereranije no kugereranya byuzuye.Kurugero, selile mumatsinda yo kuvura ugereranije na selile mumatsinda yo kugenzura,
Ni kangahe mRNA ya X gene ihinduka, iyi ni igereranya;mumibare runaka ya selile, mRNA ya X gen
Ni kopi zingahe, iyi ni umubare wuzuye.Mubisanzwe ibyo dukoresha cyane muri laboratoire nuburyo bwo kugereranya.Mubisanzwe,uburyo bwa 2-ΔΔctikoreshwa cyane mubigeragezo, ubwo buryo gusa ni bwo buzatangizwa muburyo burambuye hano.
2-ΔΔct uburyo: Ibisubizo byabonetse ni itandukaniro mumagambo ya gene yintego mumatsinda yubushakashatsi ugereranije na gen igenewe mumatsinda yo kugenzura.Birasabwa ko amplification efficensique zombi zerekanwe hamwe na gene yimbere imbere igera hafi 100%, kandi gutandukana ugereranije ntibigomba kurenga 5%.
Uburyo bwo kubara nuburyo bukurikira:
Δct kugenzura itsinda = ct agaciro ka gene igenewe mumatsinda yo kugenzura - ct agaciro ka gen imbere yimbere mumatsinda yo kugenzura
Δct itsinda ryubushakashatsi = ct agaciro ka gen igenewe mumatsinda yubushakashatsi - ct agaciro ka gen imbere yimbere mumatsinda yubushakashatsi
ΔΔct = Δct itsinda ryigeragezwa-Δct kugenzura itsinda
Hanyuma, ubare itandukaniro ryinshi murwego rwo kwerekana:
Hindura Ububiko = 2-ΔΔct (bihuye nibikorwa bya excel ni POWER)
Ibicuruzwa bifitanye isano:
Igihe cyo kohereza: Gicurasi-20-2023
