Hi research fellows,
does someone have experience performing qRT-PCR to detect antisense long non-coding RNA?
We have isolated RNA from human cells and would need a protocol to transcribe RNA into cDNA (sense and antisense transcripts). We already have the forward and reverse primer pairs which are published for detection of this AS lncRNA.
Thanks in advance for your help.
Hello Thomas,
Are you talking about a specific method for antisense lncRNAs or for lncRNAs in general? I work with lncRNAs and I usually do a single step RT-qPCR reaction, there are many kits available but we use the Qiagen QuantiFast SYBR® Green RT-PCR Kit. This work generally good for us but:
- It's based on SYBR so you may have problems of sensitivity, depending on how abundant is the transcript you want to quantify. TaqMan of course would be more selective and accurate.
- Some transcripts may be present in very low quantities, so you might need to amplify your RNA firstly to cDNA, and then quantify with qPCR (two steps).
Depending on these, you will need some optimization of your protocol.
I hope this is helpful for your question.
Hi Davide,
thanks for your fast reply. Indeed we expect very low quantities of the sense but especially the antisense transcript. Do you have a protocol how you amplify RNA to cDNA? Our first try did not work with our commonly used protocol for RNA to cDNA amplification.
Best
The point is to design a primer set to amplify the anti-sense transcript only and to avoid the sense one. You may refer to this article: https://www.hindawi.com/journals/bmri/2015/182389/
Good luck
Hi Thomas,
I'm sorry but I can't help much in this. If you want to have both the sense and the antisense to be amplified, a normal protocol of RNA to cDNA amplification should work, maybe using more RNA template and with random primers if you are not sure about the polyA of your transcript. If you want to have only the sense or the antisense, then the approach suggested above would be better!
Good luck with your work
Hi Thomas,
I work with a very low expressed antisense lncRNA, and for RNA to cDNA amplification I usually use Superscript II Kit (invitrogen) or HiCapacity kit (Applied Biosystems), starting form 1-gamma of total RNA. Since I'm sure that it has a polyA tail, I use polyA protocol, but I also tried with random primers, it works as well.
Good luck
I have used Superscript from Invitrogen and the SMART from Takara; both worked fine for me. The abundance of the transcript of interest can definitely affect your results as with any PCR regardless of the orientations. Otherwise I have read about strand-specific PCRs: an example here https://www.ncbi.nlm.nih.gov/pubmed/20300995 .
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