No amplification during RT-PCR - (Mar/16/2013 )
I wish to amplify a 2.5 kb gene from RNA. My colleague had isolated RNA, synthesized cDNA and I had amplified the gene using this cDNA previously. When I did RNA isolation and cDNA synthesis using the same RTase, I am unable to get any amplification for the same gene with all the PCR reagents same as used earlier. About 6 other different PCRs with the amplicon size of less than 200 bp (for qPCR) are working well with equal efficiency in both the cases. The starting tissue for both the case is almost the same, so I don't expect that this gene is not expressed in the tissue aliquote which I am using.
Is the larger amplicon size a problem and shall I expect that because of some problems during my RT reaction, larger RNAs have not completely reverse transcribed? Are there any modifications which can be made during cDNA synthesis to get rid of this problem?
How old is the RNA you are using? RNA is not very stable and if it is not stored properly, it can be easily degraded. With the previous cDNA, the primers you are currently using amplified your gene? Your gene doesn't have a specific temporal expression that may explain your lack of product?
If you are able to amplify additional genes from your recently produced cDNA, I would think that it is your current PCR method. Try new dNTP's, new buffer, maybe a new aliquot of your polymerase to see what happens.
Thanks for reply jerryshelly1.
It was less than a week old. If RNA is a problem, shouldn't amplification of other genes also get hampered? Yes, with previous cDNA current primers are giving brilliant amplification. No, I don't expect downregulation of the gene in the present cDNA. With the same set of PCR reagents previous cDNA is amplifying; also amplifying is the other set of genes with the present cDNA. So I don't blame the PCR reagents.
Do you have any other primers for your gene? possibly some RT-PCR primers?
Where is positive control of 2.5 kb amplicon?
if your 200bp amplicon is being amplified that means either you have problem with primers for 2.5kb fragment or your RNA is degraded.