Question

You and your lab mate, Eugene Yous, are performing expression-profiling experiments using RNA-Seq. You have extracted mRNA from a mouse liver. Both you and Eugene profile the same exact mRNA sample, but you decide to use polyT primer to make your cDNA whereas Eugene decides to use random priming. You obtain the exact same results across the genome except at one locus, the gene Ipt25. You find 250 reads map to Ipt25 whereas Eugene finds 45,000 reads mapping to Ipt25.

a. Propose an explanation for the discrepancy.

b. After scaling both data sets so that the total number of reads are identical in both yours and Eugene's experiments (e.g. RPKM), what will be the effect of the difference in Ipt25 expression on the observed expression of all the OTHER genes?

c. Suggest an alternative normalization scheme that is more appropriate for this problem

Answer 1

Please find the answers below:

Answer a: According to the information, the polyT primer used for PCR gave a smaller product whereas a random primer provided a longer product. As it is a very well known fact that the polyA site is found close to the terminal localization of the DNA, it might be possible that the polyT primer might not have been able to scan the complete target DNA sequence for amplification and hence only a part (terminal localization) of the template DNA was amplified, hence resulting in attenuated amplicon.

Answer b: Since the polyT primer was not capable of completely amplifying the whole DNA, the genes localized upstream to lpt25 genes would not be amplified and hence their expression would decrease.

Answer c: In order to normalize these expression schemes, use of a housekeeping gene such as 18S RNA, alpha tubulin or histone could be used against each of the primer. This would result in equivalent normalization of both the amplicons and give a comparative result.