Controls in Rt-qPCR - (Apr/23/2005 )

I am not sure what to add in each of these controls . Have any of you any protocols for them? Thanks in advance.


1. Negative RT PCR controls.

2. No-amplification controls (NACs). As i know this is for testing contamination of genomic DNA in the RNA sample.

3. No-template controls (NTCs).

Dear seasons,

I can only answer you 2 out of 3.

For example. when I run a diagnostic test for HSV:-
- A DNA extracted from HSV viral culture will serve as positive control.
- A NTC will a a reaction mix (containing dNTP, MgCl2, primers, Taq, buffer) without template. You can add H2O.
- negative contraol is yo add in DNa extracted from a non HSV infacted serum.

- NAC is I Don't know : ) sorry cann't help you.

Regards
Yong

negative RT-PCR control---RNA from a tissue where the gene is not expressed
No amplification control----if testing human gene, use a normal human genomic DNA
NTC----just the master mix plus water instead of RNA

SV

PS if your gDNA gives you the same sized product, you might want to redesign the primers!