We measured the expression of two genes before and after DSN normalization of the full-length cDNA library. These two genes are Ubi4, which is highly abundant, and Pr1.1, which is not. Unfortunately, the expression ratio before and after DSN normalization is very similar. One would expect the high abundant gene will become in the range as the the low abundant gene. Any suggestion? Explanations?
If I understand your experiment correctly, you have extracted the total RNA from your cells, convert to cDNA and then you have done your DSN treatment ? Then most of the DSN activity will target the ribosomal RNA (rRNA) and won't affect the messenger cDNA, even the highly expressed genes because the rRNA is representing more that 95% of the total RNA pool.
Thanks Simon. We isolated poly(A) transcripts from total RNA before the cDNA synthesis. I am assuming that this step should have filtered out the majority of rRNA, as the polyadenylated rRNAs are rare. If this assumption is correct, the DSN treatment should target highly abundant messenger cDNAs. In this case, we can expect a change in expression ratio between the high and low abundant genes. Am I getting it wrong?
Sorry, I was assuming it was bacterial RNA. Then, why are you using DNS normalization ?
I think that you should detect a change in the ratio. The other thing I wonder about is using DSN normalization of full length transcripts. This probably affect the annealing dynamics of the transcripts. How, I don't know...
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