I would like to measure the gene expression of a target gene in different tissues by means of qPCR. However, this gene has a single exon and I don't know how I can distinguish between gDNA and cDNA in my qPCR experiments. After RNA extraction, I will treat my samples with DNAses, however, it is very difficult to eliminate gDNA completely. One option would be to split the samples before RT reaction, one reaction includes enzyme (retrotranscriptase) and the other would be enzyme free. This second sample could be as a reference of DNA presence in my samples. Do you have any other ideas or suggestions? Thanks.
Hi,
the -RT control you are describing is exactly the way to do it. I always include this control lacking the enzyme for the RT-reaction. If your target gene for expression analysis via qPCR does not include intron or design of the qPCR primers in an intron-spanning way is not possible, this control is obligatory. Otherwise it is strongly recommended, as gDNA contamination of the cDNA template will result in a reduced efficiency of the PCR reaction. Be careful when handling the DNase, usually this enzyme is very sensitive/unstable, so be careful during handling and mixing.
Hope this helps & good luck with your experiment!
Hi,
the -RT control you are describing is exactly the way to do it. I always include this control lacking the enzyme for the RT-reaction. If your target gene for expression analysis via qPCR does not include intron or design of the qPCR primers in an intron-spanning way is not possible, this control is obligatory. Otherwise it is strongly recommended, as gDNA contamination of the cDNA template will result in a reduced efficiency of the PCR reaction. Be careful when handling the DNase, usually this enzyme is very sensitive/unstable, so be careful during handling and mixing.
Hope this helps & good luck with your experiment!
Hi, also as control for DNA contamination you can use your RNA sample(s) treated with RNAse. If you have amplification after qPCR it means you have DNA contamination, if not, your sample(s) is/are DNA free. Also, I agree with Gertrud for the rest.Good luck!
Hi,
If it is possible you can use other primers to control the presence of gDNA. You can design genomic specific primers (but it's a negative control). You can also use an another gene with primers spanning intron (for example the genes that will be used as control genes in your qPCR experiment). By this way you can verify that your RNA is not contaminated. Good luck
Hi,
I agree with everyone of you and I would like to add just a small suggestion. In your situation, design of specific primers for gDNA is highly recommended as Benoit said. I would further suggest to design these primers in a region very close to your gene. It would be even better use a primer inside the coding region of your target gene and the other primer upstream the TSS or wherever you want, considering to have a resulting amplicon of the same lenght as your QPCR primers amplification product. In this case you can ensure that the gDNA region of your gene is not present in your RNA extract, even if a small gDNA contamination could still be present after all the other pre-treatments. Good luck!
You may find that there are sample-specific differences in residual or DNAse-resistant gDNA and wish to make an adjustment to the Ct values following this method:
Correction of RT–qPCR data for genomic DNA-derived signals with ValidPrime
Genomic DNA (gDNA) contamination is an inherent problem during RNA purification that can lead to non-specific amplification and aberrant results in reverse transcription quantitative PCR (RT—qPCR). Currently, there is no alternative to RT(−) controls to evaluate the impact of the gDNA background on RT–PCR data. We propose a novel method (ValidPrime) that is more accurate than traditional RT(−) controls to test qPCR assays with respect to their sensitivity toward gDNA. ValidPrime measures the gDNA contribution using an optimized gDNA-specific ValidPrime assay (VPA) and gDNA reference sample(s). The VPA, targeting a non-transcribed locus, is used to measure the gDNA contents in RT(+) samples and the gDNA reference is used to normalize for GOI-specific differences in gDNA sensitivity. We demonstrate that the RNA-derived component of the signal can be accurately estimated and deduced from the total signal. ValidPrime corrects with high precision for both exogenous (spiked) and endogenous gDNA, contributing ∼60% of the total signal, whereas substantially reducing the number of required qPCR control reactions. In conclusion, ValidPrime offers a cost-efficient alternative to RT(−) controls and accurately corrects for signals derived from gDNA in RT–qPCR.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326333/
I think the simplest way to check the gDNA contamination is the use of the specific primers which can distinguish the gDNA from cDNA. In our laboratory, we use a set of primers which when bind to cDNA provide a 120 bp band on gel and when bind to gDNA provide a 1000 bp. After cDNA synthesis and DNAse treatment, using these primers you can check your samples.
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