Wonke umuntu ukhuluma ngomgomo wokuhlolwa kwe-qRT-PCR, idizayini yokuqala, ukuchazwa kwemiphumela, njll., kodwa ngicabanga ukuthi kufanele ngabelane nawe ngokusebenza kokuhlola kwe-qRT-PCR.Incane, kodwa imayelana nemiphumela.

Ngaphambi kokwenza i-qRT-PCR, sidinga ukuqonda okucacile kwe-RNA yethu kanye nezindlela zokusebenza.Phela imizamo yethu ihlose ukuthola imiphumela, kunokumane nje sizilolonge.Ngakho-ke ngaphambi kokwenza i-qRT-PCR, sidinga ukunquma izindaba ezilandelayo (ezinye zazo ezisebenza kuphela ku-SYBR).

1 Uqinisekile ukuthi i-RNA yakho ayehlisiwe?

I-NanoDrop 2000 ingathola kuphela ukugxila nokuhlanzeka kwe-RNA, kodwa ayikwazi ukubona ubuqotho be-RNA.

Inani le-RNA (RNA Intesity Number) lingabonisa ubuqotho be-RNA, etholwa uhlelo lwe-Agilent 2100 Bioanalyzer.

Umdwebo womdwebo wamanani we-RIN wamasampuli ahlukene e-RNA (eukaryote)

Kodwa-ke, amalabhorethri ngokuvamile awanayo i-Agilent 2100 Bioanalyzer.Kulokhu, singakwazi ukubona ngejeli ye-formaldehyde, kodwa imfuneko yenani eliphelele le-RNA iphezulu, ngakho indlela eshesha kakhulu ukusebenzisa ijeli ye-electrophoresis evamile.Kudingeka ukuthi ube sendaweni engenawo ama-nuclease, ngakho-ke kuyadingeka ukugeza ithangi le-electrophoresis, ibhodlela lesol, ubakaki wejeli kanye nekama ngamanzi e-DEPC.I-agarose nayo ayinayo i-nuclease (inqobo nje uma isanda kuvulwa), futhi I-Loading Buffer kufanele ivulwe kabusha ngokusemandleni, ngejeli engu-1.2%.

Qaphela ukuthi ijeli kufanele ichithwe ngokuphelele, ngaphandle kwalokho izobangela ama-inhomogeneous bands, njengoba kuboniswe kusampula 9 emfanekisweni.Uma i-voltage iphezulu kakhulu noma isebenza isikhathi eside izokhiqiza ukushisa futhi ibangele ukuwohloka kwe-RNA, ngakho-ke i-voltage nesikhathi kufanele kulawulwe ngokufanele.Ngaphezu kwalokho, ukusebenza kwejeli kungaphinde kunqume ukuthi ingabe ikhona yini insalela ye-DNA kusampula, futhi kubheke ukuthi ingabe likhona yini inani elikhulu lamabhande agciniwe emthonjeni okhiphayo.

Umfanekiso.Ukutholwa kwe-Gel electrophoresis ye-RNA

2 Ingabe uqinisekile ngokugxila kwe-cDNA yakho?

Okuhlangenwe nakho kwabafowethu abakhulu elabhorethri ukuthi i-cDNA yesistimu engu-20 ul etholwe ukuguquguquka ngakunye ihlanjululwa ngokuqondile ngo-20X, kuyilapho odade base-post-doctoral bahlanjululwa ngo-10X.Ngivame ukuncika esimweni.Ngenxa yokuthi izinga le-RNA elishiwo ngumuntu ngamunye lihlukile, izinga lokuhlehla nalo lihlukile, futhi ubuchwepheshe bokubuyisela emuva bungase bungazinzi.

Ngakho-ke ngaso sonke isikhathi uma ngithola i-cDNA ehlehlisiwe, ngizoqale ngiyihlambulule izikhathi ezingaba ngu-3, ​​bese ngisebenzisa isakhi sofuzo sokugcina indlu ukwenza i-RT-PCR, inani lemijikelezo ngokuvamile liyimijikelezo engama-25, ukuhlonza ukugxilisa ingqondo ethile, bese nginquma isici sokugcina sokuhlanjululwa.

3 Uqinisekile ukuthi iziqalo zakho zisebenziseka kalula?

Ingadlula ijika elincibilikayo le-qRT-PCR, kodwa lokhu kusabiza imali.Kumalabhorethri angenayo imali eningi, lapho ethola ama-primers amaningi, angasebenzisa i-RT-PCR evamile ukuze abone ukuthi iyiqembu elilodwa futhi ahlonze ukucaciswa kwama-primers.Uma ilabhorethri ingenayo imali, ukucaciswa kwawo wonke ama-primer kungabonakala kanye ngejiko lokuncibilika.

4 Ingabe uqinisekile ukuthi izimo zakho zokuhlola zifanelekile?

I-SYBR kufanele ivikelwe ekukhanyeni okuqinile, ngakho-ke zama ukucisha ukukhanya okungaphezulu lapho ungeza i-SYBR reagent, futhi udinga kuphela ukusebenzisa ukukhanya okufiphele ukuze uyiqedele.

Gcina i-SYBR ku-4°C.Uma isetshenziswa, phendulela ngobumnene phezulu naphansi ukuze uxube kahle ukuze ugweme ukukhihliza amagwebu, futhi ungavundisi ngamandla.

Abanye odade abancane bathanda ukudweba amamaki ebhodini le-PCR ngenxa yokwesaba ukuhlanganisa amasampula, okuyinto engalungile.Ngenxa yokuthi omaka bakho kungenzeka bathinte ukuqoqwa kwamasiginali e-fluorescent, ngokuvamile ngincoma abancane ukuthi basebenzise izincwadi zokubhalela zokuhlola ukuze basize ekukhumbuleni, njengoba kuboniswe ngezansi.

Umfanekiso.Umdwebo wokulayisha wesampula we-qRT-PCR

5 Uqinisekile ukuthi ukwenza kahle?

Qiniseka ukuthi ugqoka amagilavu, ugqoke amagilavu, ugqoke amagilavu, futhi usho izinto ezibalulekile kathathu.

Ukuze wehlise ukuchayeka kwe-SYBR ekukhanyeni, mina ngokwami ​​ngithanda ukungeza isifanekiso kuqala, njengoba kukhonjisiwe esithombeni esingezansi.Ngokusho kokuhlangenwe nakho, ukungezwa kwenani elincane lesifanekiso kungenzeka kubangele amaphutha esampula.Ngakho-ke, ukuze kuncishiswe iphutha elibangelwa ukungeza inani elincane lesifanekiso, ngivame ukuphinda kabili isampula futhi, futhi ngiphinde kabili inani lapho ngingeza isampula ukuze nginciphise inani le-H2O2 elingeziwe.

Umfanekiso.Umdwebo wohlelo lokulayisha i-qRT-PCR

Bese ulungisa uhlelo lwe-qRT-PCR kanje.

Umfanekiso.Umdwebo wokulungiselela uhlelo lwe-qRT-PCR

QAPHELA: Inqubo yokumisa idinga ukwenziwa eqhweni.

Ngemva kokwengeza isampula, namathisela ifilimu yokuvala obala.Zama ukuthi ungathinti ubuso befilimu yokuvala obala ngezandla zakho, vele usebenze usuka esikhaleni nhlangothi zombili zefilimu.Ngoba izigxivizo zeminwe zingase futhi zithinte ukuqoqwa kwezimpawu ze-fluorescent.Bese usebenzisa i-centrifuge ukusheshisa i-centrifuge amasekhondi angu-10 ngesivinini esiphansi ukuze uvimbele isampula ekulengeni odongeni.

Imikhiqizo Ehlobene:

Ikhithi ye-Cell Direct RT-qPCR

I-RT Easy II