When performing qPCR, I have noticed that cDNA made from RNA preparations treated with Turbo DNase (Invitrogen) prior to reverse transcription produce CT values that are shifted several cycles higher than cDNA made from untreated RNA. Can the DNase treatment step cause damage or loss of mRNA transcripts?
Turbo DNase in RNase free, so you need not worry about it damaging your RNA. And the shift in Ct values you observe is non-specific amplification from DNA present in your untreated RNA sample. So it is compulsory to perform DNase treatment step before Reverse transcription.... UNLESS you work with eukaryotes and design your forward and reverse primers in different exons.
Siva Ramakrishna Uppalapati Hi thank you for your response. This highlights another issue I find just with one gene of interest. I am working with RNA from human peripheral blood cells extracted using QIAGEN RNeasy kit and QIAshredder. I use oligo dT priming for my reverse transcription which should target just polyA-containing mRNA, and the qPCR primers I am using for interleukin-10 have been designed to cross exon boundaries so should not amplify from genomic DNA. (These primers have been used in previous studies never requiring the DNase step, however the samples in my current study consistently produced a product in my -RT controls which caused me to have to add the DNase treatment to the protocol). When used previously without the DNase treatment (and without any gDNA issues in negative controls) the CT of IL10 was around 30 in samples from the same cell types, in my current assays always >34
I had assumed the -RT product I was seeing was caused by contamination of my PCR setup, but was repeated when using all fresh reagents and decontaminated instruments. I can only assume then that my original RNA preps must be somehow contaminated with cDNA as opposed to gDNA?
If your NTC (no template control) is negative, then your reason should be bonafide. All the best.
Hi Harry, I would like to contribute some information, which might be helpful for your scenario. I personally avoid gDNA removal step due to many factors, which are all ending up not really in a desirable situation.
Make sure your primers are well designed across an exon-exon junction of your GOIs. As, often a primer spanning 2 exons, but within them exist a quite smaller intron hence the result is an ultimate gDNA amplification. So be sure to avoid such exons.
DNAse are usually non-specific endonucleases cleave ss, ds DNA and DNA/RNA hybrids. It is well known that any type of gDNA removal strategy would result in some loss of RNA quantity & quality. DNAse treatment is more recommended for tissue samples quite rich in DNA like the thymus, testis, spleen or if your GOI are very low copy number ones, so we prefer to do the gDNA treatment for such RNA. So for normal cases intron spanning primers are quite fine in fact.
Often due to gDNA treatment, if RNAse are there by somehow they become activated and degrades RNA. DNAse need divalent cations to work, but these cations may also induce heat-mediated RNA cleavage. More often, the inactivation temperature range for DNAse is 65 - 75 C for 10 minutes, during which you may lose some of your valuable RNA.
If DNAse treatment still giving peaks so using the probe-based assay designed across the exon junctions of your GOI is the ideal solution.
Thermofisher offers ezDNAse, which is highly specific for ds DNA only and also can be deactivated at low temperature (55 C for 5 minutes), you can try this to be more safe in terms of your RNA load in your hands....
Best Regards....
Of course such treatments can damage yor RNA and delay your cDNA at RTqPCR. I think the first step you should follow is to consider if such treatment is necessary for your samples, maybe yours have little gDNA. If you cannot avoid this treatment due to large amount of gDNA, make some pilots and use the lowest turbe enzyme quantity which give you the best performance and less RNA damage
- Badges
- Science method