RT-PCR GAPDH Variability - (Oct/18/2008 )
Hi everyone, I'm hoping you can shed some light on an issue I'm having with an RT-PCR reaction:
We don't do a lot of these, and we usually get fabricated primers and use a Hot Start PCR kit, along with a commercial first strand synthesis kit. In the past we've done RT-PCR for IL-8 mRNA and had no problems.
This time, I'm trying to do the same thing with MCP-1 (sourced from rat IEC-6 cells). We collect our nucleic acids with a standard trizol/chloroform/isopropanol protocol and use 260/280 spectrophotometry to determine concentration. then perform the first strand synthesis completely according to kit reactions, then we run the PCR reaction for 26 cycles (annealing temperature 60degC) with GAPDH as our internal control.
Normally, slight fluctuations in the GAPDH occur, but this time I'm getting a LOT of variation. We run each sample at three concentrations (.5ug, .25ug and .125ug), but I cannot get the GAPDH bands to even out -- which makes my data pretty much useless.
Is this a concentration issue? Should I increase the number of cycles and/or lower the annealing temperature? I've tried with three different sets of samples and ended up with a very similar result.
(Top band is GAPDH, bottom one is MCP-1)
RNA concentration is just one potential issue. If you can get a nanodrop to do the concentration, you will have more accurate concentration.
Check you RNA quality by running an agarose gel to see whether those samples with weak gapdh band have RNA degraded.
Make sure your samples have no DNA contamination, because gapdh have pseudogenes and its primers will amplify not just the cDNA but pseudogenes and DNA.
Make sure your PCR block has equal amplification efficiency across wells.
If you amplify gapdh with your target gene together, run them separately.
Hope that helps.