Dear Ariadne
- In essence I agree with the last commentator
- I will add to that by saying that it isn't always necessary: In essence, when you perform a standard curve to verify primer efficiency you carry out a 1:10 (high copy number) or 1:2 serial dilution of your cDNA (low copy number) to compute priming efficiency. That assay also gives you the optimal dilution of your cDNA:
- By optimal I mean that your Ct value should ideally fall between 15 cycles and 25 cycles
- For high copy number genes this might entail dilution but for low copy number genes, often even with no dilution Ct values are > 25.
- Thus, I have often used neat cDNA for low copy number genes and 1:10 for high copy number GOI and 1:100 for structural housekeeping genes
- You need a standard curve to ascertain the optimal dilution
- For high expression genes, because good data is compatible with 10-25 cycles then a series of dilutions will yield optimal data. That being so, within this permissible dilution range, select the lowest cDNA dilution to generate data:
- Lower dilutions will minimise the effect of inhibitors and also are less likely to incur problems with primer dimers (if sybr green analysis)
Hi, it is actually not necessary to dilute your cDNA if the amount of cDNA you are using is large for qPCR. It is also depending on the amount of total RNA you convert during synthesis. If you convert at 1 microgram, you get equivalent amount of cDNA and if further used for qPCR at, let's say 100 nanogram for template, out of typical 20 microlitre of cDNA product, you take 2 microlitre. No dilution required. However if you are using template at a very small amount, like currently I have been using at 10 nanogram per 20 microlitre of qPCR reaction, I have to dilute it 10 times. Without dilution, I would have to take 0.2 microlitre out of my cDNA product which I would say is too micro volume and prone to error. Further, handling volume that small will be difficult. So dilution is just to make your life easier.
Dear Ariadne
- In essence I agree with the last commentator
- I will add to that by saying that it isn't always necessary: In essence, when you perform a standard curve to verify primer efficiency you carry out a 1:10 (high copy number) or 1:2 serial dilution of your cDNA (low copy number) to compute priming efficiency. That assay also gives you the optimal dilution of your cDNA:
- By optimal I mean that your Ct value should ideally fall between 15 cycles and 25 cycles
- For high copy number genes this might entail dilution but for low copy number genes, often even with no dilution Ct values are > 25.
- Thus, I have often used neat cDNA for low copy number genes and 1:10 for high copy number GOI and 1:100 for structural housekeeping genes
- You need a standard curve to ascertain the optimal dilution
- For high expression genes, because good data is compatible with 10-25 cycles then a series of dilutions will yield optimal data. That being so, within this permissible dilution range, select the lowest cDNA dilution to generate data:
- Lower dilutions will minimise the effect of inhibitors and also are less likely to incur problems with primer dimers (if sybr green analysis)
Hi Penalva,
You have got good and informative answers already. I, whoever, would like to point out that the word "dilution" is a slight misnomer here. In fact, the concentration of the cDNA in a quantitative PCR or simply the DNA in normal PCR is the normalize the concentration of the template that you are starting with. This is important to do because this concentration has to be in symphony with the amount of other moieties used in the PCR mastermix. if the concentration is too low, the amplification may not even show up and if the concentration is too high, the results may be erroneous.
Hope this is useful.
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