I'm doing my my thesis on miRNA differential expression. A question arose when preparing for standard curve, should cDNA or its PCR product be used? When can we use each of them? Another question is that can we do qPCR for different amplicons in the same run? I think efficiencies should be same for the latter.
Whatever is most similar to the samples you are measuring is intuitively the most appropriate template for standard curve preparations. If your samples are cDNA, then the standard curve is best suited by using a cDNA mixture (made the same way your samples have been; e.g. use a mixture made of a little bit of each of your samples most suspected of expressing all of your targets of interest).
When differently-prepared template is used for the standard curve, you can expect different efficiencies of amplification even for the same target (e.g. when comparing a cDNA standard curve to a PCR product standard curve; in most cases, you will get a different amplification efficiency for Target A in each case).
There are mathematical ways to reconcile the two different efficiencies, but you can side-step the math manipulation here by using a sample type that is most like the experimental samples you are assessing.
See: http://www.horizonpress.com/cimb/v/v12/129.pdf
On your second question - yes. It is OK to test for more than 1 target per plate/run.
As long as the mastermix used for all is the exact same preparation (only differing in primers used and sample). Plate-to-plate variation is kept at a minimum using the exact same mastermix pool this way as well. Also, weighing water with each pipet adjustment is an absolute must: e.g., 23 uL on a pipet, should weigh exactly 0.023 g (of water at standard temperature and pressure) on an analytical scale. Even recently calibrated pipets are inaccurate - so weighing water to prove what volume you are delivering is key.
20 uL pipets are always 1 to 2 uL off, 100 uL pipets can be as much as 2 to 5 uL off, 200 uL pipets can be as much as 5 to 8 uL off, and 1000 uL pipets can be as much as 20 uL to 40 uL off most of the time.
E.g. you may have to set the pipet at 26 uL in order to really deliver 22 uL (as proven by weiging water). and so on...
Whatever is most similar to the samples you are measuring is intuitively the most appropriate template for standard curve preparations. If your samples are cDNA, then the standard curve is best suited by using a cDNA mixture (made the same way your samples have been; e.g. use a mixture made of a little bit of each of your samples most suspected of expressing all of your targets of interest).
When differently-prepared template is used for the standard curve, you can expect different efficiencies of amplification even for the same target (e.g. when comparing a cDNA standard curve to a PCR product standard curve; in most cases, you will get a different amplification efficiency for Target A in each case).
There are mathematical ways to reconcile the two different efficiencies, but you can side-step the math manipulation here by using a sample type that is most like the experimental samples you are assessing.
See: http://www.horizonpress.com/cimb/v/v12/129.pdf
On your second question - yes. It is OK to test for more than 1 target per plate/run.
As long as the mastermix used for all is the exact same preparation (only differing in primers used and sample). Plate-to-plate variation is kept at a minimum using the exact same mastermix pool this way as well. Also, weighing water with each pipet adjustment is an absolute must: e.g., 23 uL on a pipet, should weigh exactly 0.023 g (of water at standard temperature and pressure) on an analytical scale. Even recently calibrated pipets are inaccurate - so weighing water to prove what volume you are delivering is key.
20 uL pipets are always 1 to 2 uL off, 100 uL pipets can be as much as 2 to 5 uL off, 200 uL pipets can be as much as 5 to 8 uL off, and 1000 uL pipets can be as much as 20 uL to 40 uL off most of the time.
E.g. you may have to set the pipet at 26 uL in order to really deliver 22 uL (as proven by weiging water). and so on...
Is it necessary to check PCR efficiency when we do real time PCR with comparative Ct method for miRNA gene expression studies?
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