Simple question about LRE

[Michelle Jungbluth]

Hello,
I have what might be a simple question as to use of the LRE
analyzer, and the answer might be that I can't use it as a post-hoc
analysis method... I have qPCR data generated on a BioRad icycler with
SybrGreen chemistry, using a standard curve for quantification
purposes. Am I able to use the raw data generated from that run in the
LRE analyzer software to get (potentially more accurate) quantification
of the DNA in my samples? I am a little confused about the lambda DNA
as a calibrator... can I input my standard curve DNA quantities (not
lambda DNA, a mtDNA amplicon based on primers designed to amplify
species-specific DNA) and their respective fluorescence data as my
calibrator?

Thanks for your help,
Michelle

[Bob Rutledge]

Currently, the optical calibration function of the LRE Analyzer requires lambda gDNA to be used as a universal standard, from which an average optical calibration factor (OCF) is derived. However, an OCF can be determined manually if, as is in your case, a known quantity of target is amplified. Start by importing the profiles of your mtDNA standards as if they were normal samples, into a new Experiment database. These profiles should then be presented in the Experiment window, but their quantities will be shown as an infinity (∞) symbol, because an OCF has not yet been entered.  

To manually derive an OCF, start by entering a value for the OCF within the Experiment window, based on ~5% of the F_max of your profiles. Then adjust the OCF value until the LRE derived quantities (i.e. # of target molecules) for your mtDNA standards approach their predicted quantities. Note that the relationship between the OCF and target quantity is linear, and that increasing the OCF will decrease the LRE derived quantities, whereas decreasing the OCF will increase them.

Indeed, this illustrates nicely the universal nature of optical calibration (albeit specific to the optics of the reaction setup and qPCR instrument), such that any sample of known quantity can be used to derive an OCF. During development of LRE qPCR, lambda gDNA was chosen as a universal standard primarily due to its ubiquitous nature, which also precludes the need to prepare target-specific standards.

Hope this is of help.

Bob Rutledge