Simon Cowen

Evaluation of digital PCR for absolute DNA quantification.

The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

AbstractCirculating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. FigureComparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA

A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability.

MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.

Micromachined heated chemical reactor for pre-column derivatisation

A micromachined heated chemical reactor is presented. The microreactor consists of a sandwich of glass and silicon chips and contains a reactor channel of 50 μl volume. On-chip heating resistors supply a power of 2 W, resulting in a heating rate of 2°C/s. On-chip measuring resistors enable feed-back control of the temperature. The reactor volume makes the device suitable for derivatisation purposes in HPLC. As an example the pre-column derivatisation of amino acids with NBD-F is demonstrated, requiring 2 min of heating at 60°C, followed by HPLC separation and fluorescence detection.

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS

BACKGROUND Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. METHOD Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. RESULTS Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%. CONCLUSIONS The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.

Detection of rare drug resistance mutations by digital PCR in a human influenza A virus model system and clinical samples

ABSTRACT Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

BACKGROUND Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.