Question

NEED ANSWER WITHIN 6 HOURS

a) In quantitative PCR, what detection method would you use to quantify the threshold concentration, ct of a specific gene in a mixture. Describe the fluorescent probe that would be used and the mechanism that generates a fluorescent signal that is proportional to the number of copies in the PCR reaction. b) Describe how you could use this approach to quantify the relative concentrations of multiple, different genes in a complex mixture, using qPCR. c) How would you quantify the concentration of a specific gene in a qPRC reaction? d) What is reverse transcriptase quantitative PCR and how is it used measure gene transcription efficiency?

Answer 1

q PCR is a method to measure the exact quantify of mRNA in the samples.

There are 2 detection method used

1. Fluorescence detection method:

2. Primer or oligonucletide specific to the target.

cT is nothing but the point at which the fluorescence bevomes measurable is called Threshold concentration.

Fluorescent probe uses an intercalating dye that adds itself into Double stranded DNA. SyBR Green dye is used commonly . in every cycle number of sections of DNA is doubled leading to exponential target amplication. So the amount of fluorescence released during multiplication is directly proportinal to amplified DNA.

b. Multiple gene expression can be measured using multiplex RT- PCR / micro arrays using multiple probes.

c. By using many dilutions of the standard DNA a standard curve can be generated & concentration can be calculated against cT. The amount of DNA of interest in the sample can be calculated from the cT value.

d. RT PCR. Reverse transcriptase PCR is a diferent form of PCR that typically measures RNA expression. complementary DNA is made by reverse transcribing of RNA using enzyme reverse transcriptase and then qPCR is used to amplify and the quantified.