Hi,
I've been having some problems with my RTqPCR results. I use columns for RNA extraction and treat with DNase (in the same columns) before doing the RT. Also, when I designed the primers I made sure that they include an exon-exon junction, so that I don't get DNA amplified. When I do RTqPCR, the -RT control has amplification, but with a different melting curve than my samples (a little bit lower). It can't be primer dimers because the NTC (no template control) is negative. What could it be amplifying the -RT?
It means DNA is still present in your sample. Threat the RNA sample with DN-ase, clean RNA and retry.
Mispriming, most likely. The real question is: what are the Cq values?
If the Cq values for your -RT controls are 6 or more cycles greater than your +RT samples, then ignore this: your contamination (be it primer dimer, mispriming or whatever) contributes less than 2% of your signal, so can safely be disregarded.
There are many causes;
1. Mispriming
2. Contamination of micro-tubes, water, etc.
3. Mistake in doing DNAse treatment (specially in time).
4. Contamination during separating RNA from DNA and Pr.
It's better after RNA isolation, used 2 or 3 times from centrifuge.
Thanks for your answers.
Agnieszka Bernat How can I have DNA in my samples if my primers are designed with an exon-exon junction?
Hamed Shoorei How is it possible not to get product in the only water wells and get product in the noRT wells? If it is contamination I should have product in the water.
John Hildyard the Cq values from -RT controls aren't 6 or more cycles greater than your +RT samples.
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