小中大我做了两个多月的半定量RT-PCR,用两步法做的。结果在用housekeeping gene调PCR模板量时,怎么也得不到一致的量,是不是RT之后,用RNase-H 将cDNA单链和RNA的杂交体进行处理,驱除RNA,然后再测cDNA的浓度,再以相同的模板量进行PCR,就可以了?假如采用一步法,要定量的话,测完各个样品的RNA浓度后,以相同的RNA量进行RT-PCR就可以了?
热切盼望哪位高手帮助!
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sorry i can not type chinese now.
it does not need RNase-H digestion,beside the single strand of cDNA is different. the Pcr procedure is changeable and not standerd in dfferent time. so u should do inner control every time and caculate the ratio of target gene and housekepping gene.