Dear all,
Do I need to normalise the RNA before making the cDNA for qPCR to find out the absolute quantification in copy numbers? Why? What logic behind it when the primers amplify the DNA from the available specific DNA which was converted from total RNA.
In normalisation we measure the total RNA, converting it as cDNA, in PCR the primer specific amplicons will come. In such a case why we need to measure the total RNA before making cDNA?
Dr.Prakash S.K. The normalization of RNA makes sure that no Bias was added by pipetting since a larger RNA will give earlier Ct or Cq while lesser RNA will give a late Ct or Cq. Thus, if you fix the amount of RNA (Normalize) that you 'pip-out' in each reaction tube means you were unbiased and the results are a more near representation of the actual expression pattern.
Total RNA is not used while preparing the cDNA since it depends on the machine sensitivity and your reaction efficiency etc. Total RNA is measured to get an idea of RNA that you would use for cDNA preparation.
Hello Prakash
Absolute qPCR does not require normalization. It will measure the copy number of whatever you have incorporated in your reaction. Although, you will draw an standard curve based on using different concentrations of the standard template.
- In short no you don't
- What is important is that you make cDNA FROM EXACTLY the same amount of RNA for each biological replicate
- Normalisation is then performed by including 1-2 housekeeping genes on your plate
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