RNA is a single strand, must be transcribed to cDNA, then you can perform PCR, right?

So I need to use mRNA RefSeq instead of Genomic sequence. But I also need to choose a set of primers that will work whether I use an RNA sample or a DNA sample obtained from the patient to perform PCR? How can I make that possible?

If you want to check the gene mutation or not, you can use genomic DNA sample. If you want to check the expression of COL1A1 gene, you can use RNA sample, to make cDNA by RT, then real-time PCR

I'm not familiar with your gene, but I will assume that it is from a eukaryotic organism.

If so, all you need to do is to ensure that both primers are within a single exon.

Andrew Jenkins Yes, we will use the human COL1A1. Is there a reason why we should make sure that primers are within a single exon?

Basically, I want to be able to use the same primer pair I designed and perform PCR whether I'm using DNA or RNA samples obtained from a patient.

The coding sequence of eukaryotic genes is usually broken up into a series of shorter segments (exons) separated by non-coding DNA (introns). The introns have to be removed by RNA splicing to produce a functional mRNA.

If your primers target different exons, there will be an intron between them in the DNA and the PCR product will be much larger - potentially too large to be amplified efficiently.

Andrew Jenkins Product size must be between 400-800, however, the longest exon is only 282 bp so I can't find any primers that meet this expectation. If I include introns as well, in order to get a product size of 400-800, would it affect the detection of DNA or RNA molecules? I want to be able to perform PCR using DNA or RNA samples.

Is there any special reason the product has to be so long?

If you have to have a long product, try to find a primer pair that spans a short intron. If there aren't any short introns, you're stuck.

Andrew Jenkins As I mentioned above this is related to an assignment from one of our professors and she gave us a specific range of the product size. I understand that the intron should be short. I want to ask how short it should be.