RNA in cDNA - Can I use the same PCR primers for the production of cDNA? (Nov/17/2006 )

Hello,

For the production of cDNA from RNA: can I use the same reverse primer that I will use for the PCR later on? Or should I use dT random primer?
Thank you for your help!

Adri

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I usually use the forward primer for cDNA synthesis, then add the reverse in the PCR reaction, or P6 (random hexamer) for unspecific RT, then regular PCR reaction with fw and rev primers. If you plan to use oligo dT, make sure that your RNA is polyadenylated.
using a specific primer for CDNA synthesis will theoretically increase the specificity of your products, but it's less productive than random priming

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[quote name='lillymay' date='Nov 21 2006, 11:03 AM' post='77876']
[quote name='Adri' post='77468' date='Nov 17 2006, 06:07 PM']
Hello,

For the production of cDNA from RNA: can I use the same reverse primer that I will use for the PCR later on? Or should I use dT random primer?
Thank you for your help!

Adri
[/quote]
If you plan to use oligo dT, make sure that your RNA is polyadenylated.


Hi, thanks for your help! I isolated RNA using Trizol reagent, how can I be sure that it is polyadenylated? Should I add polyA to the RNA?
thanks ,

Adriana

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[quote name='Adri' date='Nov 22 2006, 02:09 PM' post='78066']
[quote name='lillymay' date='Nov 21 2006, 11:03 AM' post='77876']
[quote name='Adri' post='77468' date='Nov 17 2006, 06:07 PM']
Hello,

For the production of cDNA from RNA: can I use the same reverse primer that I will use for the PCR later on? Or should I use dT random primer?
Thank you for your help!

Adri
[/quote]
If you plan to use oligo dT, make sure that your RNA is polyadenylated.


Hi, thanks for your help! I isolated RNA using Trizol reagent, how can I be sure that it is polyadenylated? Should I add polyA to the RNA?
thanks ,

Adriana
[/quote]
The characteristics of your RNA must be known: for example, ~ a half of plant RNA viruses simply encode a polyA tail as part of their genome.
If your RNA is not naturally polyA tailed, you may use the reverse primer for RT, then fw and (same)reverse for PCR (it's that I wanted to point out in my previous reply, I did it and it worked after some 3 weeks optimizations!).
It is less complicated (except the optimization work), more target specific, but less productive. It also depends on the aim of your further PCR: if specific gene is to be detected only, specific primers should be fine; if more downstream applications required, you may need all RNA revers transcribed, in that case better random priming or oligodT, than primer specific PCR.

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