Aliyense akukamba za mfundo yoyesera qRT-PCR, mapangidwe oyambirira, kutanthauzira zotsatira, ndi zina zotero, koma ndikuganiza kuti ndiyenera kugawana nanu ntchito yoyesera ya qRT-PCR.Ndi yaying'ono, koma ndi zotsatira.

Tisanachite qRT-PCR, tiyenera kumvetsetsa bwino za RNA yathu ndi njira zogwirira ntchito.Ndi iko komwe, cholinga chathu ndicho kupeza zotsatira, osati kungoyeserera chabe.Chifukwa chake tisanachite qRT-PCR, tiyenera kudziwa zotsatirazi (zina zomwe zimagwira ntchito ku SYBR).

1 Kodi mukutsimikiza kuti RNA yanu sinanyozedwe?

NanoDrop 2000 imatha kuzindikira ndende ndi chiyero cha RNA, koma sichingazindikire kukhulupirika kwa RNA.

Mtengo wa RNA (RNA Intesity Number) ukhoza kusonyeza kukhulupirika kwa RNA, yomwe imadziwika ndi Agilent 2100 Bioanalyzer system.

Chithunzi chazithunzi za RIN zamitundu yosiyanasiyana ya RNA (eukaryotes)

Komabe, ma laboratories nthawi zambiri alibe Agilent 2100 Bioanalyzer.Pankhaniyi, tingathe kudziwa mwa gel osakaniza formaldehyde, koma chofunika kuchuluka kwa RNA ndi mkulu, kotero njira yachangu ndi ntchito wamba gel osakaniza electrophoresis.Imafunika kukhala pamalo opanda nyukiliya, chifukwa chake ndikofunikira kutsuka thanki ya electrophoresis, botolo la sol, bulaketi ya gel ndi chisa ndi madzi a DEPC.Agarose nawonso alibe ma nuclease (bola angotsegulidwa kumene), ndipo Loading Buffer iyenera kutsegulidwa mwatsopano momwe mungathere, ndi 1.2% gel.

Dziwani kuti gel osakaniza ayenera kusungunuka kwathunthu, apo ayi zidzachititsa magulu inhomogeneous, monga momwe chitsanzo 9 chithunzi.Ngati magetsi ndi okwera kwambiri kapena akuthamanga kwa nthawi yayitali amatulutsa kutentha ndikuyambitsa kuwonongeka kwa RNA, motero magetsi ndi nthawi ziyenera kuyendetsedwa moyenera.Kuonjezera apo, kuthamanga kwa gel kungathenso kudziwa ngati pali zotsalira za DNA mu chitsanzo, ndikuwona ngati pali magulu ambiri osungidwa mu chitsime chogawa.

Chithunzi.Gel electrophoresis kuzindikira kwa RNA

2 Kodi mukutsimikiza za kuchuluka kwa cDNA yanu?

Zomwe abale akulu mu labotale amakumana nazo ndikuti cDNA ya 20 ul system yomwe imapezedwa ndikusintha kulikonse imachepetsedwa 20X, pomwe alongo a postdoctoral amachepetsedwa 10X.Nthawi zambiri ndimadalira momwe zinthu zilili.Chifukwa chakuti mtundu wa RNA wotchulidwa ndi munthu aliyense ndi wosiyana, mlingo wa kusintha umakhalanso wosiyana, ndipo teknoloji yosinthira ikhoza kukhala yosakhazikika.

Chifukwa chake nthawi iliyonse ndikapeza cDNA yosinthidwa, ndimayimitsa kaye nthawi za 3, kenako ndikugwiritsa ntchito jini yosungiramo nyumba kuti ndipange RT-PCR, kuchuluka kwa mizunguliro nthawi zambiri kumakhala mikombero 25, kuti ndizindikire ndende yake, ndiyeno kudziwa chomaliza cha dilution.

3 Kodi mukutsimikiza kuti zoyambira zanu ndizosavuta kugwiritsa ntchito?

Itha kudutsa njira yosungunuka ya qRT-PCR, koma izi zimawononga ndalama.Kwa ma laboratories opanda ndalama zambiri, akapeza zoyambira zambiri, amatha kugwiritsa ntchito RT-PCR wamba kuti awone ngati ndi gulu limodzi ndikuzindikira zomwe zimayambira.Ngati labotale siifupi ndi ndalama, tsatanetsatane wa zoyambira zonse zitha kudziwika kamodzi kudzera munjira yosungunuka.

4 Kodi mukutsimikiza kuti zoyeserera zanu ndizoyenera?

SYBR iyenera kutetezedwa ku kuwala kolimba, choncho yesani kuzimitsa nyali ya pamwamba powonjezera SYBR reagent, ndipo muyenera kugwiritsa ntchito kuwala kocheperako kuti mumalize.

Sungani SYBR pa 4°C.Mukagwiritsidwa ntchito, tembenuzani pang'onopang'ono mmwamba ndi pansi kuti musakanize bwino kuti musachite thovu, ndipo musagwedezeke mwamphamvu.

Alongo ena achichepere amakonda kujambula zizindikiro pa bolodi la PCR kuopa kusakaniza zitsanzo, zomwe ziri zolakwika.Chifukwa zolembera zanu zitha kukhudza kusonkhanitsidwa kwa ma siginecha a fulorosenti, nthawi zambiri ndimalimbikitsa achinyamata kuti agwiritse ntchito zolemba zoyeserera kuti athandizire kukumbukira, monga zikuwonekera pansipa.

Chithunzi.Chithunzi cha qRT-PCR chotsitsa

5 Mukutsimikiza kuti mukuchita bwino?

Onetsetsani kuti mwavala magolovesi, kuvala magolovesi, kuvala magolovesi, ndi kunena zinthu zofunika katatu.

Kuti muchepetse kuwonekera kwa SYBR kuti iwonekere, ine ndekha ndimakonda kuwonjezera template poyamba, monga momwe tawonetsera pachithunzichi pansipa.Malinga ndi zomwe zachitika, kuwonjezera pang'ono kwa template kumatha kuyambitsa zolakwika zachitsanzo.Choncho, kuti muchepetse cholakwikacho chifukwa chowonjezera pang'ono template, nthawi zambiri ndimawirikiza kawiri chitsanzo kachiwiri, ndikuwonjezeranso chiwerengerocho powonjezera chitsanzo kuti muchepetse kuchuluka kwa H2O2.

Chithunzi.Chithunzi chojambula cha kutsitsa kwa qRT-PCR

Kenako konzani dongosolo la qRT-PCR motere.

Chithunzi.Chithunzi chokonzekera dongosolo la qRT-PCR

ZINDIKIRANI: Njira yosinthira iyenera kuchitidwa pa ayezi.

Pambuyo powonjezera chitsanzo, ikani filimu yosindikiza yowonekera.Yesetsani kuti musakhudze pamwamba pa filimu yosindikizira yowonekera ndi manja anu, ingogwirani ntchito kuchokera mumlengalenga kumbali zonse za filimuyo.Chifukwa zolemba zala zitha kukhudzanso kusonkhanitsa kwa ma siginecha a fulorosenti.Kenako gwiritsani ntchito centrifuge kuti muthamangitse centrifuge kwa 10 s pa liwiro lotsika kuti sampuli zisapachikidwa pakhoma.

Zogwirizana nazo:

Cell Direct RT-qPCR zida

RT Easy II