is there any experimental procedures to confirm the presence of cDNA after reverse transcription before pcr??
use the conventional pcr with house keeping gene primers
That's a tricky thing to quantify before the qPCR.
The cDNA kit only performs 1st strand synthesis, so the product is ssDNA and it should be present at the same concentration as the RNA. Indeed, these products will likely remain base paired until the melting step of the qPCR. Also, the reactions will contain unincorporated random primers, which are also ssDNA.
You can't quantify by A260 because of the RNA and the random primers. There aren't any RNA specific dye's I'm aware of to distinguish the RNA vs the ssDNA.
You might, and I'll stress *might*, be able to get evidence of cDNA synthesis by agarose gel electrophoresis. You could run out the products of a cDNA reaction made with RT and RNA, and another lane without RT, another without RNA, and another without RNA or RT and detect the products by EtBr staining or a SYBR dye.
The RT+RNA lane should have a lot of DNA/RNA hybrids, which will stain with EtBr (I think) or a SYBR dye.
The No-RT lane will have only RNA and random primers, but no cDNA. This may manifest as a difference in mobility and staining intensity.
The No RNA lane will have only random primers, which may be detectable, but which will have a very different mobility than endogenous RNA or generated cDNA.
The No-RNA & No-RT lane should look more-or-less like the no-RNA lane.
All this said, it's easier to just run the control genes as suggested above. If you're worried about a failed cDNA reaction, it's easy to run a quick test with just a few reactions. The cDNA kits are too expensive to run this kind of agarose electrophoresis step for more than a handful of samples.
I hope this can help you.
http://www.biotechniques.com/BiotechniquesJournal/2006/August/Quantification-of-cDNA-generated-by-reverse-transcription-of-total-RNA-provides-a-simple-alternative-tool-for-quantitative-RT-PCR-normalization/biotechniques-40123.html
Assuming you want to check for cDNA prior to PCR, which is specifically what you asked about, I agree with Brian Boyer's suggestion to run a gel, with the controls he listed. The other answers about use of control gene primers is an obvious part of RT-PCR, but is apparently not what you asked about.
Take genomic DNA in in one tube and cDNA in second tube, use same primers for both tubes. Be sure primers designed had intron in it. The product from CDNA will be smaller than genomic DNA, that will prove indirectly that cDNA existed.
I agree with Subhash for eukaryotic samples, but this will not work for prokaryotic samples (as prokaryotic gDNA does not contain introns). Ribogreen and Oligogreen ('oligreen') have been reported as useful fluors to measure the concentration of single-stranded DNA species such as cDNA in solution.
Well adding to the original question, i am trying to amplify a 3kb cDNA fragment usind cDNA as the template. the cDNA is prepared by using random hexamers. But the problem is i get inconsistent amplification and it does not work everytime. any inputs which may help?
thanks
You're not trying to amplify the whole 3kb in a real-time format, are you?
I did do agarose gel electrophoresis but only for cDNC and found no band and I am worrying about the next step. my cDNA kit is all done
Dear Brian,
i prepare the DNA first by using standard protocol using a cDNA synthesis kit. then use that around 0.5 uL as template to amplify the 3 kb fragment. even when i get amplification the yield is really low. And i get a lot of non specific bands of around
I'm used to using qPCR to measure gene expression using ~100-200bp amplicons. I'm not sure if qPCR can be adapted to measure such a long product, but perhaps someone else could weight in on that topic. I thought the assay lost linearity if it became too long, or that there were issues with amplification.
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