After RNA extraction, when do we do the gel electrophoresis? Besides the bands of the RNA there is a light band of a genomic DNA as well. Do we need to treat it with DNases so we can further go for cDNA synthesis? Or if we don't treat it with DNases and done with cDNA synthesis, on the next step (for real-time PCR), will SYBR green intercalate into the gDNA along with cDNA sample? Then how we can evaluate the actual expression of any gene?
Yes, you need to treat your RNA samples with DNaseI before cDNA synthesis.
As you correctly pointed out, SYBR green will bind to any double-stranded DNA present in your sample, including gDNA, which will obscure your gene expression values.
It is also very important to do noRT controls and then run a qPCR assay for your gene of interest on those samples too, along with your cDNA samples. If your DNaseI treatment was successful, you will not see amplification of your GOI in noRT samples. If you do see amplification, you will need to repeat DNaseI treatment. However, there seems to be a rule of thumb that if the difference in Ct values between cDNA and noRT control samples is more than 10 cycles, contribution of gDNA contamination is considered to be marginal.
Yes, you need to treat your RNA samples with DNaseI before cDNA synthesis.
As you correctly pointed out, SYBR green will bind to any double-stranded DNA present in your sample, including gDNA, which will obscure your gene expression values.
It is also very important to do noRT controls and then run a qPCR assay for your gene of interest on those samples too, along with your cDNA samples. If your DNaseI treatment was successful, you will not see amplification of your GOI in noRT samples. If you do see amplification, you will need to repeat DNaseI treatment. However, there seems to be a rule of thumb that if the difference in Ct values between cDNA and noRT control samples is more than 10 cycles, contribution of gDNA contamination is considered to be marginal.
Yes, there is need to treat RNA samples with DNase before cDNA synthesis and if you don't treat then they can affect your results with RT PCR.
Yes it is required to treat your total RNA prep with DNAse I. And be sure to kill the DNase enzyme after the treatment.
Yes, it is recommended to treat sample with DNAse I during RNA isolation
Of course yes, to remove complete DNA contamination from isolated RNA DNAse treatment is must.
Yes, it is necessary to treat RNA samples with DNase to minimize genomic DNA carryover that can affect your results. Always include a NRT (no Reverse Transcriptase) control into your plate to rule out the possibility to have gDNA amplified in your reaction. Also design primers that span an exon junction.
- Badges
- Science topic