The RNA extract on agarose gel shows DNA left behind so the concentration of RNA detected by nanodrop measure both. If you do the RT-PCR comparing your gene and the GAPDH would you rely on the results especially that the GAPDH results in all groups are quite close?
If your PCR assays cross an exon-exon boundary then you should only be detecting the amplified cDNA (mRNA) target. Have you tried DNase treating your RNA sample before RT-qPCR? You could always test your assay specificity by performing an RT negative control (use target RNA but no RT enzyme for the RT-qPCR). Your qPCR result for this sample should be negative even with gDNA contamination. By performing this control you will know whether you can be confident in the RT-qPCR results.
If your PCR assays cross an exon-exon boundary then you should only be detecting the amplified cDNA (mRNA) target. Have you tried DNase treating your RNA sample before RT-qPCR? You could always test your assay specificity by performing an RT negative control (use target RNA but no RT enzyme for the RT-qPCR). Your qPCR result for this sample should be negative even with gDNA contamination. By performing this control you will know whether you can be confident in the RT-qPCR results.
I'd definitely do a DNAse treatment on the remaining RNA and re-run the RT-qPCR...
I agree with Rebecca Sanders
Firstly, if the small amount of DNA is present then your primer should amplify only RNA/cDNA not DNA as Rebecca Sanders mentioned. Second thing is if you think that DNA concentration is also add on into the final RNA concentration, in that case you should use delta delta Ct method in which you have to normalized your target gene with the reference gene and then with the control sample.
You cant analyse your data on the basis of of Ct only, so use delta Ct method. The major draw back can be that you will not be able to do absolute quantification.
All the best
Dear Eman,
I agree with Rebecca. There is no need to repeat the whole experiment with DNase digestion if your negative control (cDNA without RT enzyme, practically RNA as template) shows no amplification in the RT-PCR but I would consider doing it with future samples.
Good luck!
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