Question

Question: Next generation sequencing (NGS) has been instrumental in our every-expanding pursuit of genomic knowledge. Reverse transcriptase is an enzyme uses mRNA as a template and in the presence of dNTP's makes a complimentary DNA (cDNA, still technically not a violation of the central dogma)

Part A. if i engrafted all RNA's from a cell then purified the spliced and poly-adenylated mRNA, could i use reverse transcriptase and illumine sequencing. Explain your answer

Part B. If I wanted to compare this sample to another sample (say diseased vs normal) explain how indeces help with this process. Also explain this potential approach over qRT-PCr

Please answer A and B

Nothing is misspelled or missing, it's just how the question appear on the paper.

Answer 1

Yes, purified the spliced and poly-adenylated mRNA still can be used for reverse transcriptase and illumine sequencing. reverse transcriptase reaction involve the binding of oligo dT to polyA tail of mRNA following by synthesis of first strand of cDNA. Resulted cDNA can be used either for qPCR analysis or for generation of library foe illumine sequencing.

To compare the disease vs normal, spliced and poly-adenylated mRNA sample need to purify from both type of cells (disease vs normal). Purified mRNA will be used to generate the cDNA using reverse transcription reaction. Using qPCR analysis we can analyze the expression pattern of gene of interest. This analysis may reveal up-regulation or down regulation of gene of interest in the disease as compared to normal.