We are measuring gene expression from cDNA samples prepared from bacterial RNA. Before cDNA synthesis, we treated RNA samples with DNAse to remove putative contaminating genomic DNA. However, when we use the non retrotranscribed RNA (treated with DNAse) as a template for qPCR, we obtain an amplification curve with a Ct value that falls in the same range of the Ct obtained using cDNA. We already checked the DNAse, and we saw that in the same conditions used for treating the RNA, the enzyme is capable of degrading several micrograms of a DNA sample (so the enzyme activity seems to be OK). Water controls (water instead of RNA or cDNA template) didn`t amplify, so we discarded contamination of tubes, tips, primers and qPCR kit. Have anyone encountered problems like this? If so, can you tell me possible causes?
Hi Andres
Even though the DNase seems to be working, you cannot guarantee that all DNA is removed. If you detect very low amounts (i.e. Ct > 35) in both your cDNA+ and cDNA- samples this would indicate that the reverse transcription did not work and what you are looking at is actually DNA contaminants not removed by the DNase treatment. If however you observe normal-high levels of expression this is not a likely explanation though. Are you using random primers? If so, try adding a positive control using the same samples (a house keeping gene for example). If you are using gene specific primers, try a different primer set.
I don't think primer dimers are a problem since your water controls are blank. Also these are easily distinguished from actual amplicons by looking at the melting curves. How do the melting curves look like?
Good luck
jesper
The Ct values obtained for both cDNA and RNA samples are around 18 (too high to think that first strand synthesis is not working). I observed similar results using different sets of specific primers (targeted to different transcripts). We use random primers only for the reverse transcription (included in the first strand synthesis kit purchased from LifeTech). Primers don't seem to be the problem since, as Jesper stated, water control didn't amplify. The mistery continue.... thanks for your feedback!
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