I am doing absolute quantification RT-qPCR for the beta actin gene. I start with RNA, turn this into cDNA and use that for qPCR. I am going to get a custom oligo of the PCR amplification product to use for making the standard required for absolute quantification.
This is how I got the sequence for the custom oligo. First, I input my primers into this website: http://www.bioinformatics.org/sms2/pcr_products.html along with the mRNA sequence for beta actin. The website gave me the amplified product, which is 61 bp as expected.
My question is should I use this result sequence as my oligo, or should I reverse complement it? I am wondering this because I input the mRNA sequence into the website to get the amplicon, but I am using cDNA which would be the complementary sequence. Please let me know. Thank you
Hi, generally both can be used (both will be amplified), but in absolute quantification you would like to have your standard as similar to your target as possible. Then, cDNA seems to be better choice.
However, amplification of short oligos or doublestrand products tends to be more effective, that amplification of cDNA (this is somewhat more complex material). For the best results, you should have your standard a bit longer (ideally as long as your cDNA). You may clone the whole length cDNA PCR amplify (and even isolate single strand DNA) for use as a standard. Quite lot of work - you should consider cost/benefit.
Martin
Most folks clone their "region of interest" into a plasmid to make their standard curves. It's easier and more reliable to use plasmid DNA than PCR-amplified DNA. And you are much, much less likely to run into contamination issues with plasmid compared to PCR products.
That being said, draw out a diagram of what the cDNA is compared to the original gDNA. Convince yourself where to put the primers & their relative orientations.
If you said, the double-stranded cDNA is identical to the gDNA (just no introns), then you did it exactly right.
Katie A S Burnette Thanks. What I'm saying though is that to get my region of interest, since I already know the sequence, why not just order a synthetic oligo? My plan is to just get a synthetic oligo since it would have the known amount for the standard.
Can you get the synthetic oligo as a double-stranded molecule? Single-stranded molecules will not work as a standard.
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