In support of the PREXCEL-Q Method for qPCR

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In support of the PREXCEL-Q Method for qPCR

Postby JMG » Nov 12 2011 9:49 pm

Notwithstanding the expert review of the NLR and KOD methods exploring qPCR/RT-qPCR data in the following publication:
SURVEY AND SUMMARY
Validation of kinetics similarity in qPCR
Tzachi Bar, Mikael Kubista and Ales Tichopad

Nucleic Acids Research, 2011, 1–12
doi:10.1093/nar/gkr778

It also makes the following statement regarding the "PREXCEL-Q Method" for qPCR:
Another approach to avoid quantification errors due to PCR inhibition was proposed by Gallup and Ackermann (33). First, a small amount of template is collected from all test samples and pooled. Then, the pooled sample is serially diluted and Cq is plotted versus the dilution factor, similarly to a standard curve. Since the slope of this curve describes PCR efficiency at the very early cycles, the optimal dilution for inhibition removal can be identified from the shape of the curve and applied to all test samples.

While the concept is highly robust since virtually any inhibitor can be diluted out so its effect vanishes, in practice this requires large amounts of DNA. The authors recommend dilutions by two to six orders of magnitude, which drastically reduce the sensitivity of quantification. In addition, the method assumes that all samples are inhibited to the same degree reflected by the pooled sample, which is not practical for analysis of samples where variable inhibition is expected, such as with food, water, forensic, clinical, soil and many other types of samples. Gallup’s method is implemented in Prexcel-Q software available at http://www.gene-quantification.de/download.html#gallup.


Just to clarify a few points regarding the PREXCEL-Q Method for qPCR: In practice, the method does not require large amounts of DNA, RNA or cDNA - quite to the contrary, it prides itself on being very conservative in its use of sample material; which is one of the main goals achieved by the software that provides the namesake for the approach. Further, the statements made by the authors listed above are misleading in suggesting that the PREXCEL-Q Method only uses samples diluted "two to six orders of magnitude" for analysis. This may be true of some of the standard curves that can withstand that range of dilution, but it is not true of the individually-assessed samples themselves, which are always diluted to within the early part of each standard curve - often resulting (e.g. when inhibition is absent) in using cDNA in qPCReactions at no more than 1:50 dilution since RT. The P-Q Method implores investigators to perform sample preparations that result in producing template material that is as clean, undegraded and as inhibitor-free as possible up front, and urges the investigators to explore every possible avenue towards achieving as pure nucleic acid isolates as possible when extracting from e.g. food, water, forensic, clinical, soil and other 'environmental' samples. Simple sample dilution (as the PQ approach calculates precisely and non-excessively) is another approach (in addition to good isolation/purification methods) to overcoming sample-introduced qPCR/RT-qPCR inhibition. When performed correctly (and non-excessively), sample dilution is a very effective means by which to eliminate the influence of qPCR/RT-qPCR inhibitors introduced by samples. In other words, the PREXCEL-Q Method for qPCR already assumes that researchers are starting with 'clean' samples to begin with; e.g. starting with as pure sample material as possible is understood to be a 'given' towards lessening the need for preemptive sample dilutions in the first place.

In addition, to address differential sample inhibition and to further elaborate and expand on the PREXCEL-Q Method's "Stock I idea": different representative mixtures of samples can be made from each different particular sample type, and, in cases where different degrees or types of inhibition is expected in each individual sample, the recent PQ-based method by Gardner and Gallup et. al http://www.amstat.org/meetings/jsm/2010 ... tid=306657 proposes first using the sample mixture(s) (called "Stock I") as the initial gauge by which to determine the inhibitory threshold of a group of similar-type samples, then, suggests performing a 3-point, 1:2, 1:3, 1:4 or 1:5 dilution series of each individual sample (starting within the valid dilution range as determined by the preliminary Stock I, Stock II, Stock III etc. inhibition analyses) to get a more precise measurement of each individual sample's efficiency of amplification for each particular target of interest in each sample. There is much more finesse/subtlety to this method than the above publication portrays. Most likely, this is by no fault of the above authors since expansion, evolution and application of the PREXCEL-Q Method/approach for qPCR/RT-qPCR has become more refined over the years but without the accompanying publications on its expanded methodology or descriptions thereof (although several recent publications in the U.S., Italy and Poland have used the PREXCEL-Q method with success; and the number of publications using the PREXCEL-Q Method for qPCR/RT-qPCR continue to mount). Lack of current updates on the method has largely been the result of a 2-year period of financial drought (not unknown to many of us these days). But now, since things are 'rolling' once again (due to some unexpected funding), additional detailed descriptions of the PREXCEL-Q approach, coupled to its validation by the Cy0 Method (Guescini, Sisti et.al, 2008) are underway. There is a need to stick up for this method when it is not being discussed as precisely or as fairly as it should be at this time.
Last edited by JMG on Feb 05 2012 5:51 pm, edited 1 time in total.
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Re: In support of the PREXCEL-Q Method for qPCR

Postby JMG » Feb 05 2012 1:03 pm

In support of the "Stock I" method for target amplification PCR efficiency estimations (which we proposed in the literature for the first time in 2004 in: Grubor BM, Gallup JM, Meyerholz DK, Crouch EC, et al. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs. Clinical and Diagnostic Laboratory Immunology 2004;11(3):599–607. [PubMed: 15138188] PMCID: PMC404576). Shortly after this publication we used a sample mixture as a "Stock I." Quoted below what is (in 2012) accepted as the "gold standard" for target amplification efficiency estimations in qPCR (it is what we suggested (in 2004) as the "Stock I" method (which the PREXCEL-Q program is based around):

From:
http://www.biogazelle.com/support/qBasePlus/faq 2/5/2012

"The gold standard method for PCR efficiency estimation is a serial dilution of representative template (e.g. a mixture of RNA or cDNA from your samples). Nevertheless, there are a few algorithms (from the large number out there) that seem to provide a reliable estimate of the PCR efficiency based on a single amplification curve. Importantly, the calculated results should be precise and accurate (and many algorithms fail in this respect). Hence, various papers point at the danger of using sample specific PCR efficiencies based on a single amplification curve (or even replicate measurements). The authors rather propose to average the sample specific efficiencies to obtain a target (gene) specific efficiency. This is also what Biogazelle recommends to its qbasePLUS users. In qbasePLUS, users should provide the base of the exponential function as amplification efficiency value for relative quantification if they want to correct for target specific efficiency. The base number is the E value + 1, e.g. 1.95 for 95% efficiency (E value of 0.95)..."
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