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.

4. 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 lpt25. You find 250 reads map to lpt25 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, what will be the effect of the difference in lpt25 expression on the observed expression of all the OTHER genes?

Answer 1

a. ANS:

You obtained the exact same results across the genome except at one locus, the gene Ipt25 further You found 250 reads map to Ipt25 because You has used polyT primer to start reverse transcription considerably at the 3 prime end of the transcript where mRNA possess poly-A 3' tail thereby priming done all mRNA simultaneously upon subjected to RT-PCR. Finally You got 250 reads map to Ipt25. Whereas Eugene has used random priming to generate complementary DNA by reverse transcription by raising the 5 prime transcription results in multiple tiny cDNA (distributed throughout the mRNA) fragments generation, here Eugene got nearly 45,000 reads mapping to Ipt25.

b. ANS:

After scaling both data sets so that the total number of reads are identical in both yours and Eugene's experiments; Random priming is more better than poly T priming because reverse transcription do not reach generally 5’ end of long mRNA but it is not advised to do reverse transcription with poly T primers as it may generate secondary structure further result in improper and predominantly incomplete cDNA synthesis thats why You found 250 reads map to Ipt25 except at one locus. Reverse transcription using poly-T primers enable to stop slippage of polyA by potential annealing at the 3 prime UTR/polyA junctions.