To prepare cDNA, I took the equal concentrations of RNA of both control and experiment samples. The volume of control and experiment samples during cDNA preparation was kept equal. Do I need to check cDNA concentrations of control and experiments samples once the cDNA preparation is done? My concern is that I see the difference in beta-actin levels of control and experiment samples after semi-quantitative PCR even I took the equal volume of cDNA (1ul) for both the samples.
Please give me your opinions.
Thanks in advance.
Hi,
Yes, I used to check the RNA concentration of all my samples directly after extraction and before cDNA preparation using the nanodrop.
Hi,
While the amounts of RNA used in the reverse transcription should definitely be carefully measured, I personally never check the amounts of cDNA after the reaction. Since you've encountered issues with your actin control, it might in this case be relevant to ensure that the cDNA synthesis reactions ran smoothly across all samples.
For your cDNA quantification to be meaningful using a Nanodrop OD as a readout, I would recommend getting rid of free dNTPs and remaining RNA still floating in your tubes through an RNAse treatment and an EtOH/acetate precipitation.
Hi Avinash,
we usually check RNA concentration AND quality (nano drop + gel electrophoresis) in order to exclude RNA degradation that would be still read by nano drop but would not contribute to amplification. then we assume that retrotranscription efficiency will be roughly the same in all samples (particularly if they were prepared in parallel and are the same type of sample, i.e. treatments and controls from same cell line). If the samples are of different origins you may not be able to exclude presence of inhibitors of DNA pol in some of your samples, that may account for different results, or that different cell types have different amounts of actin (think about the morphology of the cells).
The latter takes me to the second consideration: the "reference gene" you choose for normalisation of the levels of your gene of interest should be expressed at constant levels across your samples, which means that it is not influenced by the treatment you perform. If you are not sure about this, try a second reference gene (beta-2-microglobulin, gapdh, hprt, tubulins, etc).
good luck
Hi,
If you are quantifying RNA and using equal concentrations of it for cDNA synthesis, then it is not necessary to quantify cDNA. However, this holds good as long as there is no other constraint from RNA pipetting to cDNA synthesis i.e, pipetting errors, efficiency of cDNA synthesis kit between the samples etc.
Thank you, everyone, for taking out your precious time and giving me valuable advice. It definitely helped me.
Not necessarily, if you are using the same amount of RNA at the beginning. But if you need to quantify cDNA, what are you going to use as the blank? think. Is this possible?
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