SPUD qPCR Assay and PREXCEL-Q 129 SPUD qPCR Assay Confirms PREXCEL-Q Software’s Ability to Avoid qPCR Inhibition

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD ampliconcontaining plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 ± 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 ± 0.15 (at ~97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition. Introduction In recent years, although qPCR has garnered the reputation as the foremost quantitative technique for exploring gene expression, evaluating pathogen load, detecting single nucleotide polymorphisms for allelic discrimination analysis and analyzing miRNA and gene copy numbers, the quality of its performance is altered by inhibitory substances or conditions. Inhibitory substances are introduced into the tested samples by the method of isolation, the type of sample used for nucleic acid isolation, as well as other manipulations preceding qPCR (such as phenol-chloroform precipitation, sample concentrating methods and nuclease treatments) (Bustin, 2008). To minimize inhibitory biological material carryover into samples due to the method of isolation, various companies have offered different columnbased purification kits, depending on the type of sample from which the nucleic acids are to be extracted (Wilson, 1997; Bustin, 2003; Bustin, 2005; Bustin, 2008; Bustin et al., 2009). For instance, Qiagen offers several varieties of olumns for RNA, DNA or viral RNA or DNA isolation, and PAXgeneTM technology for blood samples and MO BIO Laboratories has developed a line of products which remove inhibitory material from DNA that has been extracted from a variety of biological sources. Currently, there are no simple effective solutions for high-throughput extractions of (e.g.) plant leaf DNA, and for this and other sample types, many methods require multiple steps and additional expensive materials. Older methods are laborious, and kits based on spin columns are expensive and are often not designed with high-throughput potential in mind. In addition, column-based methods often yield DNA or RNA samples that still contain inhibitory polyphenolics and polysaccharides – making such nucleic acid isolates unsuitable for PCR amplification. The challenge of eradicating qPCR inhibition has persisted as a main problem with the assay since its inception. According to a recent survey of working practices among 100 qPCR users, 94% choose to deal with inhibition by ignoring it entirely (Bustin, 2005; Nolan et al., 2006). This represents one of the most serious and persistent deficiencies in qPCR which needs to be responsibly addressed (Bustin et al., 2005; Nolan and Bustin, 2009; Bustin et al., 2009). Some of the materials capable of inhibiting reverse transcriptase (RT) and/or DNA-dependent DNA polymerase (e.g. Taq and others) have been identified, while many of them remain as yet unknown. Too much RNA and too much DNA loaded into the reactions themselves have been demonstrated to entirely shut down the RT and/ or PCR phases of the qPCR (Gallup et al., 2006). Outside of this, substances such as IgG, porphyrin, heme, fat, heparin, humic and tannic acids, polyphenolics (including tannin), dextran sulfate, Ca+2, polysaccharides and various proteins are thought to be among the known culprits of unwanted qPCR inhibition (Tichopad et al., 2004; Gallup et al., 2006; Gallup et al., 2008). Succinctly, if a target (quantification cycle) Cq value can appear anywhere from 13 to 50 on account of varying degrees of inhibition alone, it is always important to examine and/or eliminate inhibition from qPCR (Bustin, 2005; Nolan et al., 2006; Gallup et al., 2006; Bustin, 2008). Since no method is entirely effective at removing inhibitory substances from all samples, once a method of nucleic acid sample isolation and subsequent qPCR have been worked out, testing for the presence of inhibition in each sample is necessary since every sample (even from the same biological source material) can still harbor differing degrees of inhibitory material. To this end, the SPUD assay was developed (Nolan et al., 2006). The SPUD assay utilizes a synthetic amplicon based on a potato sequence in conjunction with a 6FAM-TAMRA hydrolysis probe and associated primers to amplify the SPUD sequence during qPCR (this would most likely work in a SYBR Green-based qPCR format, but it has not yet been tested as such). The SPUD amplicon is spiked in equally into all samples and standards preceding qPCR, and, in the presence Horizon Scientific Press. http://www.horizonpress.com Curr. Issues Mol. Biol. 12: 129-134. Online journal at http://www.cimb.org