Question

1.

A.) What does the Product of the RT step represent relative to gene expression in any cell or tissue?

B.) Explain the function of the different components in the RT and PCR experiments.

Answer 1

A. RT step = Reverse Transcription step.

It involves the conversion of mRNA to cDNA. The amount of a particular cDNA produced depends upon the number of the corresponding mRNA molecules present in the sample. So, if a gene is highly expressed in a cell, its mRNA and cDNA copy number would be high after the RT reaction.

If a gene is expressed at a very low level in a cell, its mRNA and cDNA copy number would be low after the RT reaction.

B. RT reaction components:

i. Reverse transcriptase = Enzyme that converts RNA to DNA

ii. dNTPs = Substrates for DNA synthesis

iii. Oligo dT = Binds to the poly-A tail of the mRNA and serves as a primer.

iv. Buffer = Provides optimum reaction conditions (pH and ions)

v. RNase inhibitor = Prevents the degradation of RNA in the sample.

vi. Water

PCR reaction components:

i. Template DNA

ii. dNTPs

iii. Taq

iv. Buffer

v. Primers

vi. Water