Kuyesera kwa RT-qPCR kumaphatikizapo kutulutsa kwa RNA ndi kuwunika kwaubwino, kubwereza mobwereza ndi masitepe atatu a qPCR, gawo lililonse lili ndi njira zambiri zodzitetezera, tidzafotokoza mwatsatanetsatane pansipa.

Ⅰ.Kuyesa kwaubwino wa RNA

Pakuyesa kwa RT-qPCR, pambuyo pomaliza kutulutsa kwa RNA, mtundu wa RNA uyenera kuwunikiridwa, ndipo kuyesa kotsatira kumatha kuchitika pokhapokha atayenerera.Njira zowunikira zimaphatikizapo spectrophotometer, Agilent gel electrophoresis, Agilent 2100 kusanthula, zomwe zimagwiritsidwa ntchito kwambiri ndi spectrophotometer ndi agarose gel electrophoresis njira yodziwira.Tiyenera kuzindikira kuti njira ziwirizi ziyenera kugwiritsidwa ntchito pamodzi kuti akwaniritse kuzindikira ndi kusanthula ndende ya RNA, chiyero ndi kukhulupirika, kuti atsimikizire kuti RNA ili yabwino.

Zogwirizana ndi RNA Isolation Kit: 

Cell Total RNA Isolation Kit

RNA yoyeretsedwa kwambiri komanso yapamwamba kwambiri imatha kupezeka m'maselo osiyanasiyana azikhalidwe mu 11min.

Animal Total RNA Isolation Kit

Kuchotsa mwachangu komanso moyenera RNA yapamwamba komanso yapamwamba kwambiri kuchokera kumagulu osiyanasiyana anyama.

Spectrophotometer:

The spectrophotometer makamaka ntchito kudziwa ndende ndi chiyero cha RNA, koma sangathe kudziwa kukhulupirika kwa RNA ndi zotsalira genomic.Pakati pawo, A260/280 ndi A260/230 ndi magawo ofunikira pakuzindikiritsa kuyera kwa RNA, ndipo kuyera kwa RNA kumatha kuzindikirika molingana ndi kusinthasintha kwamitengo yawo:

1. 1.9< A260/280< 2.1, kusonyeza kuti chiyero cha RNA ndi chabwino;A260/280<1.9, kusonyeza kuti pangakhale mapuloteni otsalira mu RNA;A260/280>2.1, zomwe zikuwonetsa kuwonongeka pang'ono kwa RNA, komwe kumatha kutsimikiziridwa ndi agarose gel electrophoresis.

2. 2.0< A260/230< 2.2, kusonyeza kuti chiyero cha RNA ndi chabwino;A260/230< 2.0, kusonyeza kuti pangakhale zotsalira za organic reagents mu RNA, monga phenols, Mowa kapena shuga.

Agarose gel electrophoresis:

Agarose gel electrophoresis assay akhoza kusanthula umphumphu wa RNA, ma genome ndi zotsalira za mapuloteni, koma sangathe kuwerengera molondola kuchuluka kwa RNA kapena kuzindikira zotsalira za organic reagents.Tengani ma tempulo a eukaryotic RNA mwachitsanzo:

1. RNA inali pansi pa agarose gel electrophoresis.Ngati panali magulu atatu okha a 28sRNA, 18sRNA ndi 5.8sRNA pamapu a gel, zikuwonetsa kuti RNA yochotsedwayo ilibe.Ngati pali chokoka chodabwitsa, izo zimasonyeza tsankho kuwonongeka kwa RNA.

2. Ngati pali gulu limodzi lowala pakati pa dzenje la guluu ndi gulu la 28sRNA, pangakhale zotsalira za DNA za genomic.

3. Ngati magulu akuwonekera mu dzenje la guluu, zimasonyeza kuti pangakhale zotsalira za mapuloteni ndi zinthu zina za macromolecular.

Ⅱ. Sinthani zomasulira

Pambuyo pochotsa RNA ikamalizidwa, iyenera kusinthidwa kukhala cDNA kuti iyesedwe motsatira, kotero kuti kusinthako ndikofunikira.Zolemba zobwerera m'mbuyo zidzayambitsidwa kuchokera pakusankhidwa kwa reverse transcriptase ndi primer:

Sinthani kusankha kwa transcriptase:

Ma reverse transcriptases amaphatikizapo AMV RTase ndi MMLV RTase.RNase H ya AMV RTase ili ndi ntchito zolimba, kutalika kwa kaphatikizidwe kakang'ono, kaphatikizidwe kocheperako komanso kukhazikika kwamafuta (42 ~ 55 ℃).Ntchito ya RNase H ya MMLV RTase ndi yofooka, kutalika kwa kaphatikizidwe ndiutali, kuchuluka kwa kaphatikizidwe ndikwambiri, komanso kukhazikika kwamafuta ndi koyipa (37 ~ 42 ℃).

Chifukwa RNase H enzyme ali ndi ntchito yonyozeka RNA Chinsinsi, MMLV ndi ofooka RNase H ntchito ayenera preferentially anasankha pa m'mbuyo transcript, ndipo pambuyo chibadwa uinjiniya, ndi matenthedwe bata la MMLV wafika pa khalidwe kudumpha.Kutenga ForegeneForeasy Reverse Transcriptase(M-MLV for reverse transcript) mwachitsanzo, ndi reverse transcriptase yatsopano yomwe imafotokozedwa mu E. coli mabakiteriya opangidwa pogwiritsa ntchito teknoloji yogwirizanitsa ma genetic.Ndi recombinant DNA polymerase yomwe imapanga chingwe chothandizira cha DNA kuchokera ku RNA, DNA, kapena RNA: DNA hybrid.Ilibe ntchito ya RNase H, kukhazikika kwamphamvu, kuyanjana kwamphamvu kwa RNA, komanso kuzindikira kwakukulu.

 

Foreasy Reverse Transcriptase(M-MLV for reverse transcript)

Kusankhidwa kwa primer:

Nthawi zambiri zoyambira za RT zimagwera m'magulu atatu: oligo dT, zoyambira mwachisawawa, ndi zoyambira za jini.Sankhani zoyambira zoyenera kuti mugwiritse ntchito molingana ndi zoyeserera zosiyanasiyana.

1. Ngati template ndi yochokera ku eukaryotic ndipo cDNA yochedwa imagwiritsidwa ntchito pokulitsa PCR, Oligo (dT) akulimbikitsidwa;Ngati kuyesa kotsatira kumangogwiritsidwa ntchito pa qPCR, Oligo (dT) ikulimbikitsidwa kuti isakanizidwe ndi zoyambira mwachisawawa kuti ziwongolere bwino zomasulira mobwerera.

2. Ngati template ikuchokera ku prokaryotes, Random Primers kapena ma jini enieni oyambira ayenera kusankhidwa kuti alembedwe mosintha.

Ⅲ.qPCR

Fluorescence quantification imafotokozedwa makamaka pakusankhidwa kwa njira zochulukira, mfundo zoyambira, kusankha kwa ROX, kasinthidwe kachitidwe kachitidwe ndi kachitidwe kachitidwe, etc.

Kusankha njira zochulukira:

Njira zochulukira zimagawidwa m'njira zochulukirachulukira komanso njira zochulukira.Kuwerengera kwachibale kungagwiritsidwe ntchito kuti azindikire zotsatira za njira zina zothandizira pa jini, kuzindikira kusiyana kwa mafotokozedwe a jini nthawi zosiyanasiyana ndi kuyerekezera kusiyana kwa jini m'magulu osiyanasiyana.Mtheradi quantification akhoza kudziwa kuchuluka kwa nucleic asidi mu HIV ndi zina zotero.Tikamayesa, tiyenera kusankha njira zoyenera zowerengera molingana ndi zomwe tayesera.

Mfundo zoyambira kupanga:

Mapangidwe a primer a qPCR amagwirizana mwachindunji ndi kachulukidwe kake komanso tsatanetsatane wazinthu.Chifukwa chake, kupanga zoyambira zabwino ndi gawo loyamba lakuchita bwino kwa qPCR.Pamapangidwe a primer, mfundo zotsatirazi ziyenera kutsatiridwa mukakumana ndi mfundo zamapangidwe oyambira:

1. Kutalika kwa chidutswa chandamale kumayendetsedwa pakati pa 100 ndi 300 bp;

2. Mapangidwe a cross-exon kuti asatengeke ndi DNA ya genomic;

3. Zoyambira zomwe zidapangidwa ziyenera kuyesedwa kuti zitheke kukulitsa bwino, ndipo pokhapokha ngati mphamvu yakukulitsa ifika pamlingo (90-110%) ingagwiritsidwe ntchito poyesa kuchuluka;

4. Kukonzekera koyamba kumakonzedwa pakati pa 0.1uM ndi 1.0uM.

Kusankhidwa kwaROX:

Pakuchulukirachulukira, ROX imatha kusintha kusiyanasiyana kwa njira, kulakwitsa kwa mapaipi kapena kusiyana kwa voliyumu komwe kumachitika chifukwa cha evaporation ndi condensation mofanana, ndikuwongolera kubwereza kwa zotsatira.Komabe, ziyenera kudziwidwa kuti kusankha kwa ROX kumagwirizana ndi chidacho.Ngati chida cha qPCR chili ndi ntchito yokonza zokha kusiyana pakati pa mabowo, sichiyenera kuwonjezera ROX;apo ayi, ikufunika kuwonjezera kuwongolera kwa ROX.Othandizana nawo ang'onoang'ono pogula ma reagents ayenera kukhala molingana ndi chida chomwe chimagwiritsidwa ntchito posankha ROX yolondola, pewani zolakwika pambuyo pake.

Kukonzekera kwa reaction system:

Mavoti a 20ul ndi 50ul amakonda.Zinthu zotsatirazi ziyenera kuganiziridwa pamene ndondomeko ikupangidwa:

1. Dongosolo lothandizira liyenera kukonzedwa ndi mpweya wabwino mu benchi yoyera kwambiri, ddH yatsopano.₂O amagwiritsidwa ntchito pa kuyesa kulikonse;

2. Kuyesera kulikonse kuyenera kukonzekeretsa NTC kuti iwonetsetse ngati pali kuwonongeka kwa dongosolo, ndipo zoyambira ziwiri zilizonse ziyenera kuchita NTC pokonzekera dongosolo;

3. Kuti muwone ngati pali zotsalira za gDNA mu template ya RNA, NRT ikhoza kukonzedwa pa chitsanzo chilichonse kuti chidziwike;

4. Pokonzekera dongosolo, tikulimbikitsidwa kuchita osachepera 3 luso kubwereza chitsanzo chimodzi;

5. Pamene template ndi cDNA, tikulimbikitsidwa kuti muchepetse nthawi 5-10 kuti muchepetse kulepheretsa kwa reverse transcription system pakuyesera kwa qPCR.Ndi bwino kufufuza kuchuluka kwa template ndi gradient, kuti mtengo wa CT ukhale pakati pa 20-30;

6. Dziwani kuchuluka kwa zomwe zimachitika, onjezerani 5-10% pamaziko a kuchuluka kwa zomwe zimachitika, ndikuwerengera kuchuluka kwa kasinthidwe ka voliyumu;

7, dongosolo lakonzedwa pogwiritsa ntchito mfundo premix, kusakaniza pambuyo centrifugation ndi kuonetsetsa palibe thovu;

8, Momwe mungathere kusankha zothandizira zogwiritsira ntchito.

Zogwirizana ndi RT-qPCR Kit