I will suggest reading of an interesting article

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonaspathosystem.

https://www.nature.com/articles/srep44905

Yes, your first step is correct. Firstly, you should design the specific primer for that gene and then run the RT-qPCR workflow.

To do this, you should align the primer to the genome sequence to find whether the primer is specific.

Good luck!

Dear Noyce:

Q: To validate the quantity of one transcript from RNA seq data by RT-qPCR, Do I use a specific gene primer in the RT-qPCR workflow to isolate only this transcript sequences for the PCR step.

A: I would suggest you follow these steps:

1) Determined the assembled mRNA (cDNA) sequences of your target gene and use the sequences to design the target gene-specific primers,

2) Make sure that your designed primers were from the conserved region of the gene coding sequences. You may want to do multiple sequence alignment among the assemble RNA sequences with other CDS of the same genes.

3) Isolate total mRNA from the target tissues expressing your gene of interest and construct cDNA library,

4) Validate the designed primers (in step 1 and 2) by conducting PCR using cDNA library (in Step 3) as the DNA template. You may need to optimize the PCR conditions to suit your primers (i.e optimum Tm may be determined by using Gradient PCR).

5) You may also want to PCR using cDNA library (in Step 3) as the DNA template and the internal control gene specific primers. You will use this as internal control for quantification (RT-qPCR is not an absolute quantification method but it is a relative quantification by comparing it to internal control).

6) Once optimized PCR conditions for the target gene and the internal control gene have been determined, do the RT-qPCR analysis and determine the relative quantity of your target RNA.

I hope these steps answer your questions. Good luck.

If you didn't get the answer what you are looking for would you please clarify what do you really want to know:

1. How to design a gene specific primer for qRT-PCR and primer design considerations OR

2. The kind of reverse transcription strategy: Oligo dT / random primers/ gene specific primer?

Cheers,

Shajahan