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Should You Dilute cDNA for Real-time PCR? -- Ask TaqMan® Ep. 11 by Life Technologies

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Published on Feb 25, 2013

Download the Relative Gene Expression Workflow PDF here: http://bit.ly/1b3UKCF
Submit your real-time PCR questions at http://www.lifetechnologies.com/askta...
In this video, examine whether or not qPCR end-users doing gene expression should dilute cDNA prior to running samples. This video also looks at the possible need to dilute RNA prior to reverse transcription. This takes into consideration a host of scenarios including final reaction volume, expression level of genes under study.
It's true that a large percentage of gene expression researchers dilute their cDNA prior to running reactions. Some do a 1:3 dilution, others say 1:5 is better, while at least a few individuals insist that a 1:10 is best. Playing devil's advocate are a whole host of researchers who will gladly inform you that diluting cDNA at all is a waste of time and perfectly good, reagent-grade H2O!
So who's right and who's . . . not? Well, like so many things in life and science, it depends. To see what I mean, let's talk for just a second about the top reasons why some people choose to dilute cDNA.
First concern: high-concentration cDNA might have a negative effect on PCR, especially if it contains inhibitors carried over from the initial RNA isolation step. Mmm . . . yes and no. It's true that inhibitors can have an impact on PCR. But cDNA tends to be fairly dilute, and so inhibiting compounds are rarely introduced into PCR at sufficient quantities to cause problems.
That said, if the original RNA sample is really dirty, or contains lots of residual chemicals from the RNA prep such as Ethanol, it's likely that the reverse transcription step itself won't be successful. So failed real-time reactions? Often not caused by cDNA being dirty, but rather to an unsuccessful RT that produced little or no cDNA in the first place! That's why RNA should always be squeaky clean.
I should mention: reverse transcription buffers can inhibit PCR, especially if added to reactions at greater than 20% of the final volume. So If you aren't diluting cDNA, don't add more than, say, 5 uL of the RT reaction to a 25 ul reaction.
There is a second reason people dilute cDNA: the fear that too much cDNA will cause the housekeeping gene -- especially if it's a very high expresser like 18s -- to appear so early during cycling that an accurate baseline can't be set
I agree, this is a real concern, and probably a good reason for diluting. Just be sure not to dilute so much that your target gene Cts drift into the mid-30s or later, since this can causes problems of its own. One final note: users always have the option of limiting how much RNA goes into the initial RT reaction.
To help you determine how much RNA you should use, please visit LifeTechnologies.com and download a copy of the Relative Gene Expression Workflow Guide. Step 9 contains step-by-step instructions on how to empirically ensure that you're using an appropriate amount of template. This document also covers most other aspects of gene expression experiments.

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