Hi there,
I need to validate a series of reference genes (i.e. housekeeping genes) for my qPCR assay and I need to construct a standard curve for each primer pair to check for efficiency. I am using range of snail embryo tissue samples, as well as a snail adult tissue sample.
Due to the limited quantity of RNA from the embryos, I would prefer to use the adult tissue (of high RNA yield) to conduct the standard curves. However, I am planning to use both embryo and adult tissue samples for validating my primers, but it's likely the adult tissue samples will be excluded from the comparison of expression data.
Thus, my question is: would it be acceptable to use cDNA from the adult tissue to examine primer efficiency (e.g. constructing standard curves) as long as the sample is tested (and included in primer validation experiments), but not used in the final analysis?
I believe it is ok to use whenever the target gene is the same in both.
But to develop standard curve, more dilutions may be needed to cover the probable lower RNA concentrations found in Embryo samples.
You could just use a PCR product for your standard curve, since the overwhelming bulk of reactions in a PCR will use PCR products as their template anyway.
Just PCR up the amplicon, gel extract it so you know it's a single amplicon species, spec it and use the amplicon length to work out molecules.ul-1, then do a standard curve from maybe...10^9 molecules per well down to 0.1 molecules per well.
It doesn't matter what you use as your starting material to generate that amplicon, since sequence is sequence.
My preferred material for doing this is plasmid DNA. There are several suppliers (I use GenScript who can synthesise your target sequences and clone them into a plasmid, which they supply to you as DNA. Putting several different target sequences into the same plasmid is no problem and doesn't cost much more. You can then make a dilution series of the plasmid for you dilution series, and you can calculate the exact number of plasmid copies from the DNA concentration and the molecular weight of the plasmid.
The plasmid will cost you several hundred Euros, but you only need one and you'll have enough to last you forever. Note, however, that the concentrated plasmid is very 'hot' and is almost as good a contamination source as a PCR product. I do my first dilutions in a non-PCR laboratory and only bring it into the sample extraction lab when it's down to about 107 copies/µl.
Regarding John's suggestion of using PCR products; I have reservations as my experience with this approach has not been good. I think this was due to primer-dimer. Once formed, primer dimers amplify extremely efficiently and they can outcompete the authentic reaction. But that's just my impression. Others may have better experiences.
And finally: if your PCR doesn't cross any intron/exon junctions, you could use genomic DNA from snails. I don't recommend using RNA or cDNA for standard curves. In the case of the former, the efficiency of conversion to cDNA becomes a confounding variable, and in the latter it's challenging to estimate the concentration(s).
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