Hi all,
I am doing cell fractionation and RT-qPCR to assess the enrichment of some specific transcripts in different cell compartments.As usual, I choose like gapdh or Rpl genes as internal controls for normalizing the data, which means: all raw Ct values were first normalized to gapdh Ct, then treatment groups were normalized to Ctrl group. This was done for all the fractionation samples (cyto/nuclear/nascent RNAs). But I am afraid that this kind of normalization would impair the real enrichment since internal control genes also have preferential localization difference between cyto/nuclear. I was also advised that normalization should be done to choose different internal controls for either cyto or nuclear, such as: choose gapdh for cytoplasmic control while Neat1 for nuclear control, as those would be the right comparisons.
Also for the actinomycin D treatment experiments to assess transcript stability, internal controls with different treatment time points could also show degradation. Which I am not so sure how to normalize data to best present the real scenarios for transcripts' stability properties.
Please advise and share your experience about this issue.
Many thanks and best,
Lu
Hi Lu, sometimes it is very difficult to know which reference genes should be used. It is possible that you will have to empirically test a few candidates beforehand to make sure that they do not fluctuate under your conditions. Then you will know that your normalization is accurate.
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