impact of varying RNA concentrations on cDNA synthesis and RTPCR

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sitara
sitara's picture
impact of varying RNA concentrations on cDNA synthesis and RTPCR

Dear All
I wanted to know few things
Will there be variation in the ct values in the real time pcr if cDNA synthesised from different concentrations of RNA are used.
For example:
sample labelled as A, 1 microgram of RNA used for cDNA synthesis 
sample labelled as A, 0,4 microgram of RNA for cDNA synthesis

when using the cDNA from the above two ways (varying RNA concentration from a single sample) will here be variation  in real time PCR?
                         
Its very urgent can anyone help me please

Ivan Delgado
Ivan Delgado's picture
The simple answer to your

The simple answer to your question is yes. Since it is not trivial to quantify the amount of cDNA synthesized by the RT reaction, it is ideal to use equal amounts of RNA when synthesizing cDNA for qPCR. The assumption here is that equal amounts of cDNA will be synthesized if you use equal amounts of RNA, which while not true is a close enough approximation. 

Of course one way to get around this is to run a control qPCR assay using a housekeeping gene in each of your cDNAs and use that to control the amount of amplification for your genes of interest. In that case, the difference in the amount of RNA used is not that critical. In other words, as long as you normalize the expression of your gene of interest to the expression of a housekeeping gene in each one of your cDNAs, then you do not need to worry about the amount of RNA used to make your cDNA.

sitara
sitara's picture
 Thank you for the

 Thank you for the information. I had already done qPCR with cDNA prepared from different concentration of RNA, but even though normalising with the reference gene, there is around 0.7 ct cycle difference. 

The results are attached

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Ivan Delgado
Ivan Delgado's picture
Having a Ct difference of 0.7

Having a Ct difference of 0.7 when you are setting up identical qPCRs is a result of things like uneven pipetting or variations in the true concentration of your RNAs/cDNAs, not the qPCR itself. You may want to make sure you are pipetting your solutions correctly, possibly maybe by increasing the volumes you dispense.