QPCR with genomic DNA from blood - (Jan/20/2016 )

Hi

Im trying to design an experiment at the moment to look at the difference in expression of three genes between trained and untrained athletes.

Im not looking for copy number just expression of the gene relative to a housekeeper.

So for example, after the experiments are run i can state that relative to beta-actin the untrained athletes express 4 times less of GENE A than the trained athletes.

I am able to get a blood sample from each of the participants and extract genomic DNA from the blood.

Now i am attempting to design my QPCR experiment.

1.Can i use genomic DNA in my QPCR (Syber green) reaction?

2.When designing the primers do i need to design them within an exon to avoid really long intronic sequences?

3.Is there any recommended housekeeping genes used when dealing with gDNA?

ideally i would like to be able to get RNA from the blood and make cDNA, however it is often 24/48 hours after it being drawn before i receive the blood.

Any ideas or comments would be gratefully received.

Roddy

If you want to measure gene expression, then you need mRNA.  Using gDNA will just tell if the gene is present or no.  I prefer taqman probes over sybr green for me its more accurate. For the blood drawing is better to use edta (purple cap tubes), it will preserve better the cells, then extract the PBLs with Ficoll-Paque PLUS, I used the Accuspin tube for a better separation (but you don't have to). Once you have the pbls isolated, extract the RNA (my favorite kit is qiagen uneasy , digest with DNase1,, check rna quality,  then convert to cDNA (2 steps so you can check all genes).  Always do a RT reaction and a RTminus reaction to make sure you only have rna. PBls can be store at -80C for month or even years and you can keep extracting good quality rna. good HK genes could be beta actin, gapdh.  

Thanks so much for all of your advice.

Ill get everything ordered in and give it a go. I think deep down i knew that i couldn't use gDNA but was hoping to avoid the hassle of an RT step.

Thanks again.