Hello, everyone
In my current real-time PCR assay, I saw the beta-actin was not stable in the LPS-stimulation cells for longer time. It seems the internal control gene-beta-actin is not always work well. So I am wondering to change an internal control gene, which I found many researchers used 18S RNA. At the same time, I wonder whether the 18S RNA could be reversed-transcriptioned by using oligo-dT? Because 18S RNA do not has polyA tails, do I need more RT-primer specially for it?
Thanks for answer my question.
Just use random hexamers for RT or preferably combine random hex with oligo-dT. Or try GUSB, HPRT or GAPDH or other house-keeping genes as reference genes (beta-actin has an enormous amount of splice variants and is usually highly expressed, so it is not the best reference gene anyway).
Just use random hexamers for RT or preferably combine random hex with oligo-dT. Or try GUSB, HPRT or GAPDH or other house-keeping genes as reference genes (beta-actin has an enormous amount of splice variants and is usually highly expressed, so it is not the best reference gene anyway).
Hi
As mentioned by you already that the rRNAs do not have poly A tail ...it will be better to use either random hexamers/nonamers or primers specifically for rRNA...
bye
Cannot add to the above
Random hexamers are how you reverse transcribe species of RNA other than mRNA which by definition lack a 3' Poly A tail
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