I would like to check the cDNA after the stem loop RT-PCR reaction done.
In the normal RT-PCR, the cDNA can check by B-actin amplification. But stem loop RT-PCR is different. So can anyone tell me, how to check cDNA before the real time PCR will be performed.
thank you
pannapa pinweha
If the qPCR works, then the cDNA is fine.
If the qPCR doesn't work then either the miRNA you're looking at is not expressed, the assay for it doesn't work, or the RT didn't work.
There is no direct way to visualise or measure the cDNA, so you have to rely on a multiplexed housekeeper. In miRNA stem-loop RTs I have successfully used up to 12 stem-loop probes in one RT reaction, which I then dilute and test in individual qPCR assays. At least 2 of the probes are against housekeeping genes (at least one of the same class as my target - i.e. miRNA - and normally also an abundant one, e.g. snRNA or snoRNA).
Hope it helps!
Before going to qPCR, you can do the semi quantitative PCR with the cDNA prepared through the stem-loop RT primer, then check the product on 3% Agarose gel to see the desired size (~65 bp) of the microRNA of interest. To see such small product you need to stain/ post stain the gel with ethidium bromide.
In "normal" reverse transcription, you would get representation from all species of RNA if you use random primers (e.g. random hexamer/nonamer). Beta-actin usually works because it's abundant and thus easy to detect. I guess you are looking for miRNAs
If you're using specific primers, then Jannatul's suggestion may work, but the cDNA will be difficult to see for a couple of reasons:
1. It is single-stranded (apart from the stem-loop part). This makes dye-binding more inefficient.
2. It is small in size, so less dye can bind per molecule, which means you need a large number of molecules or a very sensitive reader to visualize them.
Of course, if you could easily do that, then there would be no need to run real-time PCR to look for your product!
What you could do is run the qPCR on a few of your samples and be sure to include samples with both high and low starting concentrations of RNA and a moderately abundant miRNA target. miR-16 is very popular, provided you included its stem-loop primer in your RT. Hope this helps!
Were you able to perform conventional PCR with the cDNAs for miRNA? If so, could you describe the step-by-step analysis used?
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