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Background: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility., Methods: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield., Results: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3' end of the primer. In addition, having ≥4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased., Conclusions: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for ≥4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence.

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology., (© 2013 American Association for Clinical Chemistry.)

Compromised RNA quality is suggested to lead to unreliable results in gene expression studies. Therefore, assessment of RNA integrity and purity is deemed essential prior to including samples in the analytical pipeline. This may be of particular importance when diagnostic, prognostic or therapeutic conclusions depend on such analyses. In this study, the comparative value of six RNA quality parameters was determined using a large panel of 740 primary tumour samples for which real-time quantitative PCR gene expression results were available. The tested parameters comprise of microfluidic capillary electrophoresis based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5'-3' difference in quantification cycle (Cq) and HPRT1 3' Cq value based on a 5'/3' ratio mRNA integrity assay, the Cq value of expressed Alu repeat sequences and a normalization factor based on the mean expression level of four reference genes. Upon establishment of an innovative analytical framework to assess impact of RNA quality, we observed a measurable impact of RNA quality on the variation of the reference genes, on the significance of differential expression of prognostic marker genes between two cancer patient risk groups, and on risk classification performance using a multigene signature. This study forms the basis for further rational assessment of reverse transcription quantitative PCR based results in relation to RNA quality., (© The Author(s) 2011. Published by Oxford University Press.)

Context: Short stature has an incidence of three in 100 in children. Reliable molecular genetic testing may be crucial in the context of beneficial disease management. Deletions spanning or surrounding the SHOX gene account for a significant proportion of patients with idiopathic short stature (ISS) and allied disorders, such as Leri-Weill dyschondrosteosis., Objective: Several shortcomings of current strategies for copy number profiling of the SHOX region prompted us to develop an improved test for molecular diagnostics of the SHOX region., Design and Results: We introduced a quantitative PCR (qPCR)-based copy number profiling test, consisting of 11 amplicons targeting clinically relevant regions, i.e. the SHOX gene and regulatory regions. To ensure an optimal sensitivity and specificity, this test was validated in 32 controls and 18 probands with previously identified copy number changes. In addition, 152 probands with SHOX-associated phenotypes were screened, revealing 10 novel copy number changes., Conclusion: This highly validated qPCR test supersedes other approaches for copy number screening of the SHOX region in terms of reliability, accuracy, and cost efficiency. In addition, another strong point is the fact that it can be easily implemented in any standard equipped molecular laboratory. Our qPCR-based test is highly recommended for molecular diagnostics of idiopathic short stature and allied disorders.

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.

The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.

Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments., Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement., Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

Basal cortisol and ACTH levels have previously been shown to be elevated in highly trained athletes, whereas the ACTH response to ovine CRH has been reported to be diminished compared to that in nonathletic controls. Naloxone, a nonselective opioid receptor antagonist, is known to stimulate ACTH and cortisol secretion. The mechanism of this response is thought to be via increased hypothalamic CRH secretion. The aim of this study was to examine basal and naloxone-stimulated levels of hypothalamic-pituitary-adrenal axis hormones in male athletes. Ten highly trained male athletes and 10 nonathletic controls took part in the study. Peripheral venous blood was sampled for cortisol, ACTH, CRH, and arginine vasopressin (AVP) for 2 h before the administration of 20 mg naloxone, i.v., and 15, 30, 45, 60, 90, and 120 min after naloxone treatment. Body mass index was significantly lower in the athletes (P < 0.001). Basal (prenaloxone) ACTH levels were higher in the athletes (P < 0.05), whereas levels of cortisol, CRH, and AVP were similar in both groups. After naloxone treatment, there was a significantly greater rise in ACTH in the athletes (P < 0.02). There was also a trend for the cortisol response to be greater, which was not statistically significant (P < 0.07). Although in both groups, peripheral CRH rose after naloxone treatment (P < 0.005), a rise of similar magnitude occurred over the 2-h period before naloxone (P < 0.0001). Plasma AVP did not change significantly after naloxone treatment. Neither the plasma cortisol level at baseline nor the body mass index correlated significantly with the ACTH or cortisol response to naloxone. The presence of an enhanced ACTH response to naloxone is evidence that central opioid tone may be increased in highly trained athletes. However, there is no associated suppression of the hypothalamic-pituitary-adrenal axis, and basal ACTH levels are raised, without any detectable change in peripheral plasma CRH or AVP. An additional factor (other than CRH) that stimulates ACTH secretion may be released after naloxone administration.

The 14CO2- glycylcholate breath test (also called the bile acid breath test) was performed in a group of 42 normal young volunteers (group A), a group of 25 elderly subjects in apparently good health (group B) and a group of 22 hospitalized geriatric patients presenting with weight loss (group C). The 95 percentile value of the cumulative 14CO2 excretion at the third and the sixth hour in group A was taken as the limit for normal values for 14CO2 excretion. Using these criteria 56% of group B subjects and 50% of group C patients were considered abnormal at the third hour, whereas at the sixth hour these percentages were 56% and 54%, respectively. Repetition of the bile acid breath test after antibiotic treatment in the hospitalized group suggested that bacterial overgrowth in the small intestine was responsible for the abnormal 14CO2 breath test in the elderly persons. However, the large number of abnormal tests in healthy elderly people, not complaining of any gastro-intestinal discomfort, indicates that bacterial overgrowth may remain asymptomatic and that an abnormal test does not necessarily mean that the symptoms of a patient are to be ascribed to this finding.