Biophys Techniques: Southern Blotting, PCR, RT-PCR

[TytoAlba95]

Homework Statement

During transgenesis, the location of genes and their number integrated into the genome of the transgenic animal are random. It is often necessary to determine the copy number of genes and their tissue specific transcription. The following are the possible methods used for the determination:
a. PCR
b. Southern blotting
c. Reverse transcriptase PCR
d.Western blotting

Relevant Equations

Ans: Southern blotting and RT-PCR

I have problem understanding these techniques with reference to the question:
a. PCR
b. Southern blotting
c. Reverse transcriptase PCR
d.Western blotting

My attempt:
d. Western blotting is not the right method here because it doesn't detect DNA or RNA transcript.
b. Southern blotting can be used to determine the copy number of the insert in the animal genome.
a. PCR can detect the presence of the gene but cannot be used to detect the copy number.

I don't understand how RT-PCR detects the tissue specific transcription level.

 

[TeethWhitener]

Hint: what are the substrate and product of reverse transcriptase?

 

[TytoAlba95]

Hint: what are the substrate and product of reverse transcriptase?

The substrate is mRNA and product is cDNA.

Could you please explain briefly how the expression level is determined by using RT-PCR?

My understanding:
RT-PCR will simply convert all types of RNAs into cDNAs (using a oligo-dT primer) then (I don't know how the single stranded cDNA is converted into dsDNA) using a set of two gene specific primers the ds-cDNA will be amplified.

But to understand the expression level I think one needs to have a control too(if so, can you please elaborate on the control).

 

[TeethWhitener]

The substrate is mRNA and product is cDNA.

Could you please explain briefly how the expression level is determined by using RT-PCR?

My understanding:
RT-PCR will simply convert all types of RNAs into cDNAs (using a oligo-dT primer) then (I don't know how the single stranded cDNA is converted into dsDNA) using a set of two gene specific primers the ds-cDNA will be amplified.

But to understand the expression level I think one needs to have a control too(if so, can you please elaborate on the control).

Yes, you’re right. Simply using RT-PCR without any quantitation will give you a binary yes/no for whether a gene is being transcribed, but won’t give you the expression level. However, you can combine RT with qPCR to get quantitative results. Here’s an overview:
https://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

 

[TytoAlba95]

This link also provides a clear overview on qPCR.