I've read that I need cDNA standard curves to test primer efficencies.
I'd like to know if I could use genomic DNA instead, since I've got too many different conditions from which different cDNAs have been synthesized.
In the end, depending on the efficencies, I've read that I may use either the Double Delta or the Pfaffl method to quantify the relative expression of genes.
Hi Diana,
Good question, you could, but its not ideal. You want to see how efficient your primers are with your samples, or a similar sample that was extracted and isolated in the same way. There may be different contaminants, detergents or compounds present depending on sample preparation that changes your primer efficiency, which this should be controlled. A probably unlikely issue with well designed primers, but still possible, is gDNA has introns that your primer might anneal to, while cDNA does not. This can be assessed by the melt curve though.
And it would be good for you to use the Pffafl method instead of the double delta. Good luck!
Typically not. Most folks studying gene expression are using primers that amplify a target region that is spanning an intron. The difference in product size from gDNA vs. cDNA would mean you can't do a fair comparison of amplification efficiency.
The only way this would be possible is if:
1. Your primers amplify the exact same product in gDNA vs cDNA (not ideal since it's much harder to rule out contamination/incomplete DNAase treatment).
2. Your DNA extraction is extremely high-quality
3. All of your genes of interest are from the same part of the genome (e.g. all nDNA, no mtDNA or cpDNA)
4. You are doing a relative expression-level study (not exact copy number).
Most folks clone their target region of interest as a cDNA into a plasmid.
Good luck!
Hi! I agree with Katie A S Burnette . In addition, one alternative method instead of cloning is design gBlocks™ Gene Fragments (Integrated DNA Technologies, Inc) with your amplicon sequences. With this approach, the fragment designed will have a known number of copies per µL, which can be used for the construction of standard curves and, consequently, absolute quantification of the number of copies/reaction in samples analyzed through qPCR.
Best regards!
The gold standard method for analysis of relative gene expression is using reference genes to normalize gene expression. I recommend you to read MIQE guidelines: DOI: 10.1373/clinchem.2008.112797.
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