qPCR
known human pseudogenes made available by Gerstein’s lab at Yale University23.
Figure 1
Probing sensitivity to genomic DNA of RT-PCR assays using ValidPrime. The intron spanning RPL7 assay and
ValidPrime, which targets a non-transcribed sequence
present in exactly one copy per haploid human genome, are used to amplify purified human genomic DNA in the dPCR OpenArray platform. A two-fold dilution series is
performed across the 64-well subarrays. Comparison reveals that the RPL7 assay, despite being designed to span an
intron, generates more PCR product from the genomic DNA than the ValidPrime
chambers, while with 8,000 chambers 10 from 11 copies can be separated. With qPCR it is challeng- ing to separate two from three copies20. Recently Henrik Laurell pointed to a problem in RT-qPCR that can be approached with dPCR21. Analysing gene expression mRNA is reverse transcribed into cDNA and quantified using qPCR. Since tran- scripts are copies of the genome the RT-qPCR assay may also amplify genomic DNA that has not been removed during the processing of the sample. Current recommendation to deal with the problem is to design qPCR assays spanning introns. The intron-spanning primers will be close to each other in the transcript and readily amplified, while they are far apart on the genomic sequence and will not give rise to PCR product. True, at least for genes that have introns. But, Henrik Laurell says, many genes have intron-less pseudo genes that are ampli- fied even when intron spanning primers are used. In fact, the number of intron-less pseudo genes of a gene in the genome may be large and highly vari- able among species. For example, the commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has 62 pseudogenes in human and 331 in mouse22. These pseudogenes may contribute with substantial background. The sensitivity of RT-qPCR assays optimised for the quantification of cDNA to genomic DNA can be tested with dPCR. Dilution series of genomic DNA with an intron-spanning RFPL assay and ValidPrime (Figure 3) reveals the RFPL assay is picking up more DNA signal than ValidPrime, which amplifies one sequence copy per haploid genome. Obviously, the intron-spanning RFPL assay is picking up signal from intronless pseudo- genes in the genome. Indeed, presence of pseudo- genes is a major complication in RT-qPCR and for serious quantitative studies it is good practice to experimentally test the importance of genomic contribution to the RT-qPCR signal. It is also advisable to consult the excellent database of
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The sequence discrimination is enhanced in dPCR by the partitioning of the sample into the large number of reaction containers, which effec- tive reduces the background level of related sequences. Consider a somatic mutation present in one out of 1,000 copies. With conventional probe-based qPCR we would not be able to detect the mutation because of insufficient discrimina- tion. However, in dPCR the reaction chamber that happens to contain the mutated sequence will have substantially lower background of the wild-type sequence and the mutation is readily detected. This advantage was employed already in the pioneer paper by Vogelstein for early detec- tion of a mutant ras oncogene in the stool of patients with colorectal cancer10, and later to quantify EGFR alterations in lung cancer patients. The same approach has been to detect non-cultivatable pathogens against excessive backgrounds25, and to probe individual environ- mental bacteria for viruses26. The improved dis- criminative ability of dPCR has also been employed for analysis of foetal DNA in plasma27, and for sensitive quantification of minimal resid- ual disease in chronic myeloid leukemia28. An esoteric dPCR/qPCR application is tomography to study the spatial distribution of transcripts in samples including gradients of transcripts within individual cells29.
More extensive dilution, such that reaction chambers rarely contain more than a single tem- plate molecule, confers additional advantages to dPCR, including more extensive multiplexing. In conventional qPCR multiplexing is in practise lim- ited to some four targets because of competition for reagents of the parallel reactions that compro- mises sensitivity and accuracy, and by crosstalk between detection channels when simultaneously quantifying multiple signals1. Clonal amplification obliterates these complications since there is no competition between reactions and there is no crosstalk. In fact, clonal amplification allows for an even higher degree of multiplexing using com- binatorial detection based on multiple probes bind- ing per target30,31. dPCR is potentially also less sensitive to inhibition. While inhibitors may delay amplification affecting the Cq values in qPCR lead- ing to erroneous concentration estimates, they do not obliterate product formation and therefore do not influence the count in dPCR. Clearly, digital PCR is a forthcoming technique that is rapidly becoming established in the field of nucleic acids analysis. Still, the wider spread of the
Drug Discovery World Fall 2011
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