I was advised to use it with my reverse RTPCR specific primer... Should I use it in combination with the primer mix from the kit, or should I use only the reverse primer and futher supplement the reaction with MgCl and dNTPs?
Thanks in advance!
Andreia
I have been using this kit with consistent success, however, I've mostly reverse transcribed very highly expressed RNAs. Presumably you are using specific primers because your gene is very lowly expressed.
Part of the reason this kit works so well is that the RT primers provided with it are a mixture of oligo-dT and random hexamers. If you mix the kit primers with your specific primer, you loose the advantage of using a specific primer to only amplify your intended RNA of interest. Just be aware that with specific primers, you loose the ability to use reference genes to normalise.
I would not change the MgCl and dNTP concentration in the mastermix - simply adjust your primer volume to compensate for the volume of kit provided primers you would add.
Dr. Petra,
I am about to use the quantitect reverse transcription kit which describes that 'Incubate for 15 min at 42°C. In some rare cases (e.g., if the RT-PCR product is longer than 200 bp or if analyzing RNAs with a very high degree of secondary structure), increasing the incubation time up to 30 min may increase cDNA yields.' My RT-PCR products length ranging from 200-750bp. If I increase the incubation time up 30 min, is it possible to amplify the 750 bp length product. Could you please share your experience/opinion on this?
Dear Panneerselvam,
I've always used 30 min anyway.
One thing to mention though - are you planning to use the 750 bp PCR product in RNA expression profiling? If so this is not a good idea, as amplification efficiency will be lower than for your shorter products and amplification efficiency is very important in accurate quantification. If you are simply making cDNA for other purposes, 750 bp shouldn't be a problem.
Thank you very much for the information Dr. Petra. I will take your suggestion :)
Dear Andreia , I am using this kit from last five years and i had not faced any problem . The beauty of this kit is gDNA wipe out buffer, so you need not to use DNAse treatment
Regarding your question , i would suggest you to use both kit primer as well as your gene specific primer. If the expression of your gene is low , your gene specific primer would help to synthesis specific cDNA of your gene of interest.
rest of the aliquot you can use for amplification of other gene.
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