I am trying to obtain cDNA from RNA samples from 5 different tissues. Currently, I am doing the following:
1ug RNA + random oligomer (50uM) + water followed by 10mins at 70 degrees C. Then I add 5X buffer, dNTPs, RNAse inhibitor and the AMV enzyme. I also have the negative controls.
I then do a PCR with my primers for the gene of interest alongside positive controls with a primers for a housekeeping gene. I am consistently getting a very faint band with my experimental primers. Also, for some of these samples I'm not getting a band with either set of primers or indicating that I may not have any cDNA to begin with.
I've been told to try the RNA extraction again and let the pellet dry fully during ethanol precipitation. Aside from this, what else could I do to increase my cDNA yield? Thank you
I agree with checking on the quality of your RNA - if there is a lot of degradation then you may have problems amplifying fragments of interest. Another route may be to change up the cDNA amplification kit you are using, I am not familiar with the one you mention above. I do know however that there are kits that give a great cDNA yield with minimal work, e.g. Quanta qScript Supermix. I use this for generating qPCR products with a low RNA input, as little as 10ng.
Another route (if necessary) is to amplify your cDNA using a specific pool of primers. There are kits out there for this purpose, whether for RNAseq or qPCR with very lowly expressed transcripts. I have not used these but have looked into them as I thought I might need to do this.
I can give you further information if needed but I hope that helps. Good luck!
I think you are facing degradation of RNA and you are using degraded RNA for reverse transcription. Just check your RNA band and its position in agarose gel. If the lower size of band is formed, you can assume that it may the reason of degradation of RNA. Similarly you can check the cDNA length. If the cDNA formed from your degraded RNA, You will not get actual result or faint amplification. You can use gene specific primers for cDNA preparation. It may resolve your problem.
10mins at 70 degrees C in the water leads you to significant degradation of your RNA. You didn't need this step. Simply mix all components (RNA and oligos and then all others) and incubate this mixture at 37-42 degrees.
What I miss in your protocol are two things, first I miss a inactivation step of your RT enzyme at the end of the reaction, normally 10 minutes at 68 degrees. RT inhibits your PCR reaction.
After this step an RNAseH treatment will improve your downstream PCR dramatically, especially for GC rich targets. RNA-DNA hetroduplexes have a very strong binding, much more then DNA-DNA duplexes, RNAseH removes the RNA strand, keeping single stranded cDNA.
Superscript III from Thermofisher is a good cDNA kit, RNAseH is included.
Hi Kunal
all the above given answers were correct.
I agree with the fact that your RT-PCR reaction is not well balanced.
RT: 1-6 ug of RNA + primers 0,2 ug + water. incubate 5 min 65 deg. and then on ice. Total volume 10 ul
cDNA: RT mix, containing RNasin-AMV etc. incubation 2 hrs 43 degrees.
PCR: PCR mix + RT mix: 40+10 ul
I should use a RT-PCR control.
Hi Kunal,, I am sure, you have all the right answers,, from my side , just focus first on getting a good quality RNA. Analyze for integrity and check the level of A260/A280 for purity. Some times long term preserved tissues have problems of low yield RNA , if this is the case than i am afraid you will continue experience the same problem.
Good luck
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