Troubleshooting RT-PCR - (Jan/02/2008 )
Hi there
I have been struggling with an RT-PCR for a few months now. Starting material is blood RNA. I only get a faint amplification along with a large number of non-specific(shorter and longer) bands. I have tried RT with gene specific as well as oligo dT primers. Oligo dt is slightly better than Gene Specific primer. I have tried all possible combinations of MgCl2, dNTP and primer concentrations. Still the amplification is very weak, not enough to be eluted. It is 2.4kb fragment. I have tried oridinary Taq and one specific for long PCRs. Doesn't work at all with ordinary Taq, gives the faint amplification with the other one. Tried varying denaturing, annealing and extension temps and times, tried touch down..... The target is a low copy gene. Any suggestions are welcome, Somebody please help me ..............
I have been struggling with an RT-PCR for a few months now. Starting material is blood RNA. I only get a faint amplification along with a large number of non-specific(shorter and longer) bands. I have tried RT with gene specific as well as oligo dT primers. Oligo dt is slightly better than Gene Specific primer. I have tried all possible combinations of MgCl2, dNTP and primer concentrations. Still the amplification is very weak, not enough to be eluted. It is 2.4kb fragment. I have tried oridinary Taq and one specific for long PCRs. Doesn't work at all with ordinary Taq, gives the faint amplification with the other one. Tried varying denaturing, annealing and extension temps and times, tried touch down..... The target is a low copy gene. Any suggestions are welcome, Somebody please help me ..............
Are you sure you can PCR directly from RNA using Taq?
'Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA'
http://nar.oxfordjournals.org/cgi/content/abstract/20/7/1487
i believe you need an extra step prior to PCR.
Do you have a cDNA clone of the gene you are trying to do RT-PCR with? Perhaps trouble shooting the PCR with a cDNA clone would be easier, then you can move on to the more difficult task of getting the PCR to work with low abundance template.
I have been struggling with an RT-PCR for a few months now. Starting material is blood RNA. I only get a faint amplification along with a large number of non-specific(shorter and longer) bands. I have tried RT with gene specific as well as oligo dT primers. Oligo dt is slightly better than Gene Specific primer. I have tried all possible combinations of MgCl2, dNTP and primer concentrations. Still the amplification is very weak, not enough to be eluted. It is 2.4kb fragment. I have tried oridinary Taq and one specific for long PCRs. Doesn't work at all with ordinary Taq, gives the faint amplification with the other one. Tried varying denaturing, annealing and extension temps and times, tried touch down..... The target is a low copy gene. Any suggestions are welcome, Somebody please help me ..............
HI,SIBB,
Please confirm that you have done it in this way:
Retro-transcrip RNA into cDNA using oligo(dT)/random primers;
Amplify your target gene using the new cDNA as template.
If it still dose not work, then try a gradient PCR.
Good luck!
I have been struggling with an RT-PCR for a few months now. Starting material is blood RNA. I only get a faint amplification along with a large number of non-specific(shorter and longer) bands. I have tried RT with gene specific as well as oligo dT primers. Oligo dt is slightly better than Gene Specific primer. I have tried all possible combinations of MgCl2, dNTP and primer concentrations. Still the amplification is very weak, not enough to be eluted. It is 2.4kb fragment. I have tried oridinary Taq and one specific for long PCRs. Doesn't work at all with ordinary Taq, gives the faint amplification with the other one. Tried varying denaturing, annealing and extension temps and times, tried touch down..... The target is a low copy gene. Any suggestions are welcome, Somebody please help me ..............
HI,SIBB,
Please confirm that you have done it in this way:
Retro-transcrip RNA into cDNA using oligo(dT)/random primers;
Amplify your target gene using the new cDNA as template.
If it still dose not work, then try a gradient PCR.
Good luck!
Tried all that, no luck!
Of course! Only after reverse transcription reaction the PCR was done.
What enzymes are you using? (both RT and DNA polymerase)
Have you considered nested PCR and how much cycles are you using?
you should try to make 3 rounds of PCR. increase the number of PCR cycles to 35.
rna-preparation
cDNA synthesis (oligodT primer, random hexamer-primers or both, or either gene-specific primers) with good quality reverse transcriptase
first PCR-round with specific primers
second PCR round with nested or seminested primers
third PCR--round with nested primers
if you do it this way, you will get tons of PCR product, even if your transcript is expressed very weak. noraml taq-polymerase should work
maybe you shoud test the quality of your RNA and/or cDNA with a highly expressed transcript (GAPDH e.g.)