Please suggest the optimum concentration of cDNA for RT PCR run. Does diluting the cDNA 1:10 or 1:100 help in increasing amplification, specially for low expressing target genes?
The reason to do a dilution study (different dilutions of the cDNA) for RT PCR (which can either mean "reverse transcription PCR" or "real-time PCR"; not clear in your question) is to find out whether or not your cDNA preparation inhibits the PCR when used too concentrated. If you add too much cDNA to the PCR, you will not get amplification - even for targets like 18S rRNA. Too much sample added to the PCR is like throwing too much wood on a fire: you smother the reaction. This is either caused by sheer template inhibition of the e.g., Taq polymerase, or by the inhibitory potential of [elements of] the cDNA prep itself on the PCR. A cDNA titration is always in order for every PCR study as a preliminary exercise. There is no 'one-size fits all' in these matters - given the different operators, tissues/cells, RNA isolation methods and reagents used. The MIQE Guidelines is also a very useful thing to read as a preamble to all of this:
http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwiFk7KilZnKAhXEGh4KHeWzA1EQFggfMAA&url=http%3A%2F%2Fwww.rdml.org%2Fclinchem.2008.112797v1.pdf&usg=AFQjCNEpeGD8fMyk0NjotTPjaD0XeeiBuw&sig2=5cxSVK5OfX-r4Gxm_53hzg&bvm=bv.110151844,d.dmo
If one shuts the PCR reaction off (or severely inhibits it) by adding too much sample, then no target, no matter how abundant or how rare, will be able to amplify.
Dear Mansi
First you have to set up the dilution of cDNA and look what concentration is good for your target gene and endogenous gene. Dilution can be start from 100ng, 10ng, 1ng and 0.1 ng. If you are using human blood as your subject then 20 ng will be the good concentration for the reaction. My personal experience says that most of the genes show good Ct near 10-20ng concentration if not then something wrong with your primer or that can be low expression gene.
All the best
Hi,
the concentration of cDNA depends the expression level of the target genes, it is difficult to give a very special concentration. in my oppion, you can try to do Real Time PCR using the different concentration of cDNA.
Hi Mansi, I agree with above answers. Best to figure it out for your samples using a dilution serials of the same cDNA sample. I performed qRT-PCR on unicell alga Chlamydomonas and my magic concentration was 1 ng.
The reason to do a dilution study (different dilutions of the cDNA) for RT PCR (which can either mean "reverse transcription PCR" or "real-time PCR"; not clear in your question) is to find out whether or not your cDNA preparation inhibits the PCR when used too concentrated. If you add too much cDNA to the PCR, you will not get amplification - even for targets like 18S rRNA. Too much sample added to the PCR is like throwing too much wood on a fire: you smother the reaction. This is either caused by sheer template inhibition of the e.g., Taq polymerase, or by the inhibitory potential of [elements of] the cDNA prep itself on the PCR. A cDNA titration is always in order for every PCR study as a preliminary exercise. There is no 'one-size fits all' in these matters - given the different operators, tissues/cells, RNA isolation methods and reagents used. The MIQE Guidelines is also a very useful thing to read as a preamble to all of this:
http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwiFk7KilZnKAhXEGh4KHeWzA1EQFggfMAA&url=http%3A%2F%2Fwww.rdml.org%2Fclinchem.2008.112797v1.pdf&usg=AFQjCNEpeGD8fMyk0NjotTPjaD0XeeiBuw&sig2=5cxSVK5OfX-r4Gxm_53hzg&bvm=bv.110151844,d.dmo
If one shuts the PCR reaction off (or severely inhibits it) by adding too much sample, then no target, no matter how abundant or how rare, will be able to amplify.
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