DNA Free RNA - Figuring out contaimination issues (Oct/05/2007 )
Hey,
I'm wondering how to exactly go about setting up my controls for DNA free RNA and testing for DNA contamination.
Do I simply run two reactions in parallel one with RT one without RT. With Primers for my housekeeping gene. If it shows up that the RT- reaction has bands for the genome DNA product we know we have contamination.
Can I only run the RT- reaction with the primers for the genome DNA housekeeping gene? Or do I need to run the RT+ and RT-
Is there any other methods to confirm your RNA is not contaminated?
Cheers,
Chris
Have you tried running in a gel to visualise it? you can use absorbance method as well but it might be as accurate only.
Did you use DEPC water and also RNAse free zap (Ambion)? very useful!
You can design a set of primers for some gene that has an intron in the middle.
By comparing the genomic and cDNA sequences you'll know the difference you'll get when you pcr.
If your RNA pcr shows a band of the same size as the genomic DNA pcr then your sample is contaminated...
By comparing the genomic and cDNA sequences you'll know the difference you'll get when you pcr.
If your RNA pcr shows a band of the same size as the genomic DNA pcr then your sample is contaminated...
it sounds good idea, but using an DNasa and runing a parallel reaction (with same oligos) without RT would be enough.
Running a gel to visualize DNA is not enough because you can have small amount of DNA not easily seen in a gel but enough to amplify.
By comparing the genomic and cDNA sequences you'll know the difference you'll get when you pcr.
If your RNA pcr shows a band of the same size as the genomic DNA pcr then your sample is contaminated...
it sounds good idea, but using an DNasa and runing a parallel reaction (with same oligos) without RT would be enough.
Running a gel to visualize DNA is not enough because you can have small amount of DNA not easily seen in a gel but enough to amplify.
sorry...
can u have further explanation of tis step??
thanx...