It depends on two factors: do you need absolute of relative quantification? If absolute, it also depends on the level of accuracy you need. While it is possible calculate the number of template copies if you start with plasmid DNA, the truth is that even linearized DNA will not behave exactly as cDNA in your reaction (though it is close!). Briefly, the stability of double stranded DNA and RNA / DNA heteroduplexes are different. While this is only important in the first few rounds of PCR, when your original template constitutes the majority of target, it is important nonetheless, especially if you are often quantifying low quantities of target. The most accurate way to do this is to produce RNA target molecules, using in vitro transcription. In this way spectrophotometry can be used to calculate the number of RNA template copies. This is not a quick and dirty process, however. I would only recommend it if the absolute accuracy of results is of primordial importance. For most applications, I agree with Alejandro, linearized plasmid would suffice.

On the other hand, if you are looking for relative target abundance between two or more sources, using cDNA dilutions is completely acceptable.

You can use plasmid DNA, but have to linearize it first. See e.g. PMID 20221433 (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2832698&tool=pmcentrez&rendertype=abstract)

It depends on two factors: do you need absolute of relative quantification? If absolute, it also depends on the level of accuracy you need. While it is possible calculate the number of template copies if you start with plasmid DNA, the truth is that even linearized DNA will not behave exactly as cDNA in your reaction (though it is close!). Briefly, the stability of double stranded DNA and RNA / DNA heteroduplexes are different. While this is only important in the first few rounds of PCR, when your original template constitutes the majority of target, it is important nonetheless, especially if you are often quantifying low quantities of target. The most accurate way to do this is to produce RNA target molecules, using in vitro transcription. In this way spectrophotometry can be used to calculate the number of RNA template copies. This is not a quick and dirty process, however. I would only recommend it if the absolute accuracy of results is of primordial importance. For most applications, I agree with Alejandro, linearized plasmid would suffice.

On the other hand, if you are looking for relative target abundance between two or more sources, using cDNA dilutions is completely acceptable.

I do agree that in vitro transcribed RNA will be the best standard. Plasmid DNA not suitable for SYBR green qPCR. Plasmid DNA is ok for TaqMan. For cDNA, exact how efficient the RNA is reverse transcribed is not known, meaning that accuracy on the copy number might be affected.

Hope someone is still watching this question... Does anyone have a reference that speaks for/against the use of plasmid DNA for establishing standard curves in qPCR? We (and I'm certain many others) have good data showing that the dynamics of plasmid vs cDNA are very different, but I'm wondering if anyone has bothered to publish this.

Dr. Laflamme, I did not find a publication which addresses your question. Does your data reflect different DNA polymerase activity on cDNA vs DNA? Or perhaps that there are inhibitors in the cDNA?

Hi Lauren (Please call me Mark :-) - None of our data is publication worthy at this point, nor to do we expect to bring it up to that level, but.. We have data showing better performance of certain polymerases than others, but this is not consistent between assays. For example, we have 9 validated diagnostic assays on our scope that all use TaqMan MGB technology. 6 work best with the TaqMan Universal Mastermix and 3 work best with the TaqMan GeneExpression kit. Using Plasmid vs cDNA to calculate reaction efficiency, we were surprised to see that efficiency was quite different. Perhaps this could be due to inhibitors, but I did find one paper that speaks of conformations issues as well.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0029101

Although the paper investigates circular vs supercoiled plasmid, it also stands to reason that the stability of the initial cDNA/RNA hybrid is different than that of the DNA/DNA hybrid... This being said, perhaps we're splitting hairs at this point. We're going to keep looking at this and see if it is worth pushing.

Cheers,

Mark

Dear Dr. Tan Chee Wah

Why is it not possible to use plasmid DNA with SYBR Green?