FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols.

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.

Loss of CD73-mediated actin polymerization promotes endometrial tumor progression.

Ecto-5'-nucleotidase (CD73) is central to the generation of extracellular adenosine. Previous studies have highlighted a detrimental role for extracellular adenosine in cancer, as it dampens T cell-mediated immune responses. Here, we determined that, in contrast to other cancers, CD73 is markedly downregulated in poorly differentiated and advanced-stage endometrial carcinoma compared with levels in normal endometrium and low-grade tumors. In murine models, CD73 deficiency led to a loss of endometrial epithelial barrier function, and pharmacological CD73 inhibition increased in vitro migration and invasion of endometrial carcinoma cells. Given that CD73-generated adenosine is central to regulating tissue protection and physiology in normal tissues, we hypothesized that CD73-generated adenosine in endometrial carcinoma induces an innate reflex to protect epithelial integrity. CD73 associated with cell-cell contacts, filopodia, and membrane zippers, indicative of involvement in cell-cell adhesion and actin polymerization-dependent processes. We determined that CD73-generated adenosine induces cortical actin polymerization via adenosine A1 receptor (A1R) induction of a Rho GTPase CDC42-dependent conformational change of the actin-related proteins 2 and 3 (ARP2/3) actin polymerization complex member N-WASP. Cortical F-actin elevation increased membrane E-cadherin, ß-catenin, and Na(+)K(+) ATPase. Together, these findings reveal that CD73-generated adenosine promotes epithelial integrity and suggest why loss of CD73 in endometrial cancer allows for tumor progression. Moreover, our data indicate that the role of CD73 in cancer is more complex than previously described.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

Variability of the reverse transcription step: practical implications.

BACKGROUND: The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability. METHODS: We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay. RESULTS: RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed. CONCLUSIONS: Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

CGRRF1 as a novel biomarker of tissue response to metformin in the context of obesity.

OBJECTIVE: Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention. METHODS: CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and Western blot. RESULTS: CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveals that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease. CONCLUSION: CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.

The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.

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.

Molecular clustering based on ERα and EIG121 predicts survival in high-grade serous carcinoma of the ovary/peritoneum.

Assessment of estrogen receptor (ER) expression by immunohistochemistry has yielded inconsistent results as a prognostic indicator in ovarian carcinoma. In breast and endometrial carcinomas, panels of estrogen-induced genes have shown improved prognostic capability over the use of ER immunohistochemistry alone. For both breast and endometrial cancers, overexpression of estrogen-induced genes is associated with better prognosis. We hypothesized that analysis of a panel of estrogen-induced genes can predict the outcome in ovarian carcinoma and potentially differentiate between tumors of varying hormonal responsiveness. From a cohort of 219 women undergoing ovarian cancer surgery from 2004 to 2007, 83 patients were selected for inclusion. All patients had advanced stage ovarian/primary peritoneal high-grade serous carcinoma and underwent primary surgical debulking, followed by adjuvant treatment with platinum and taxane agents. The expression of ERα and six genes known to be induced by estrogen in the female reproductive tract (namely EIG121, sFRP1, sFRP4, RALDH2, PR, and IGF-1) was measured using quantitative RT-PCR. Unsupervised cluster analyses were used to categorize patients as high or low gene expressors. Gene expression results were then compared with those for ER immunohistochemistry. Clusters were compared using χ(2) analyses, and Cox proportional hazards models were used to evaluate survival outcomes. The median follow-up time was 38.7 months (range: 1-68). A cluster defined by EIG121 and ERα segregated tumors into distinct groups of high and low gene expressors. Shorter overall survival (OS) was associated with high gene expression (HR 2.84 (1.11-7.30), P=0.03), even after adjustment for other covariates. No difference in ER immunohistochemistry expression was noted between gene clusters. In contrast to other hormonally driven cancers, high expression of ERα and the estrogen-induced gene EIG121 predicts shorter OS in patients with high-grade serous ovarian carcinoma. Such a biomarker panel may potentially be used to guide management with estrogen antagonists in this patient population.

S100A4 mediates endometrial cancer invasion and is a target of TGF-beta1 signaling.

The molecular mechanisms of endometrial cancer invasion are poorly understood. S100A4, also known as FSP1 (fibroblast-specific protein 1), has long been known to be a molecular marker of fibrosis in a variety of different fibrotic diseases of the lungs, liver, kidney, and heart. We demonstrate here that increased expression of S100A4 is associated with advanced stage endometrial cancer and decreased recurrence free survival. To verify the essential role of S100A4 in invasiveness of endometrial cancer, S100A4 expression was downregulated by RNAi in HEC-1A cells, which resulted in undetectable S100A4 protein and significantly decreased migration and invasion. Owing to the established connection between TGF-beta1 and S100A4 induction in experimental models of kidney and liver fibrosis, we next examined whether TGF-beta1 could also regulate S100A4 in endometrial cancer cells. TGF-beta1 stimulated endometrial cancer cell migration and invasion with a concomitant increase in S100A4 protein. Induction of S100A4 was associated with the activation of Smads. TGF-beta1-mediated endometrial cancer cell motility was inhibited by S100A4 siRNA. In aggregate, these results suggest that S100A4 is a critical mediator of invasion in endometrial cancer and is upregulated by the TGF-beta1 signaling pathway. These results also suggest that endometrial cancer cell invasion and fibrosis share common molecular mechanisms.

Enhanced estrogen-induced proliferation in obese rat endometrium.

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

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.

Hypomethylation-induced expression of S100A4 in endometrial carcinoma.

Expression of various S100 genes has been associated with clinically aggressive subtypes in a variety of different cancers. We hypothesized that S100A4 would be overexpressed in endometrial carcinoma compared to benign endometrium. Quantitative real-time RT-PCR (qRT-PCR) was used to quantify the mRNA level of S100A4 in benign endometrium (n=19), endometrioid adenocarcinoma (n=87), and non-endometrioid tumors (n=21). Immunohistochemistry was used to verify the results of qRT-PCR and to assess protein localization. Possible mechanisms of S100A4 gene regulation were also examined. S100A4 was overexpressed in the grade 3 endometrioid tumors, uterine papillary serous carcinoma, and uterine malignant mixed müllerian tumor. Expression in grade 1 and grade 2 endometrioid tumors was comparable to that of normal endometrium, which was quite low. Expression was significantly higher in stage III and IV tumors compared with stage I. By immunohistochemistry, S100A4 was expressed in the tumor cell cytoplasm of poorly differentiated tumors, but was not detected in normal endometrial glandular epithelium. In benign endometrium, S100A4 expression was confined to stromal cells. S100A4 was not regulated by estrogen or progesterone, and its expression in tumors was not significantly correlated to estrogen receptor or progesterone receptor content. However, methylation of the S100A4 gene was detected in benign endometrium and grade 1 tumors with low S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 mRNA and protein expression showed no methylation of the gene. These methylation results were verified in endometrial cancer cell lines with differential baseline levels of S100A4 protein. These results suggest that hypomethylation is an important mechanism of regulating the expression of the S100A4 gene. These results support the emerging concept that hypomethylation may play a role in the upregulation of genes during later stages of tumorigenesis.

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens.

A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.

Ingested (oral) IFN-alpha represses TNF-alpha mRNA in relapsing-remitting multiple sclerosis.

In a phase II trial in relapsing-remitting multiple sclerosis (RRMS), patients ingesting 10,000 IU, but not 30,000 IU, interferon-alpha (IFN-alpha) showed fewer gadolinium enhancements at months 5 and 6, along with decreased proinflammatory tumor necrosis factor-alpha (TNF-alpha) protein secretion. Therefore, we examined MxA mRNA induction and TNF-alpha mRNA repression after 100, 300, 1,000, 3,000, and 10,000 IU doses of ingested IFN-alpha in 24 RRMS patients to determine the optimal dose for future clinical trials in MS. Maximal TNF-alpha repression occurs at 100, 1,000, and 3,000 IU. These data provide new optimal doses for additional clinical studies using ingested IFN-alpha in MS.

Identification of a novel estrogen-regulated gene, EIG121, induced by hormone replacement therapy and differentially expressed in type I and type II endometrial cancer.

PURPOSE: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. EXPERIMENTAL DESIGN: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. RESULTS: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium. CONCLUSIONS: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.

Molecular diagnosis of cutaneous diseases.

OBJECTIVES: To provide an update on the molecular procedures used increasingly in the study and diagnosis of a variety of dermatologic malignancies and inflammatory disorders and to explore the potential use of these techniques in clinical dermatology. Herein, we review assays such as G-banding, fluorescence in situ hybridization, comparative genomic hybridization, and spectral karyotyping in conjunction with the polymerase chain reaction and DNA microarrays. DATA SOURCES: PubMed was searched for published articles on molecular diagnosis and dermatologic diseases. STUDY SELECTION: All English-language studies were selected if they provided useful methodologic information or highlighted the usefulness of molecular techniques. DATA EXTRACTION: Only methodologic and qualitative information was extracted. DATA SYNTHESIS: The information was synthesized into 2 sections: one describing the principles of different molecular diagnostic techniques, and the other highlighting the contributions of molecular diagnostic techniques to the understanding and diagnosis of several dermatologic diseases. CONCLUSIONS: A basic understanding of the principles of molecular diagnostic techniques is crucial for the practicing dermatologist to benefit from the increasing number of molecular diagnostic articles appearing in the literature and potentially to apply these methods in clinical practice.

Estrogen regulation of the cytochrome P450 3A subfamily in humans.

This study examines the possible role of estrogen in regulating the expression of the human CYP3A subfamily: CYP3A4, CYP3A5, CYP3A7, and CYP3A43. To accomplish this goal, mRNA was quantified from human livers and endometrial samples, and total CYP3A protein levels were evaluated by Western immunoblot analysis of the liver samples. The human endometrial samples were from premenopausal and postmenopausal women. The premenopausal endometrium was either in the proliferative or secretory phase, whereas for the postmenopausal endometrium samples, the women had been treated with either a placebo or estropipate, an estrogen substitute. After analyses, CYP3A4 mRNA was shown to have lower hepatic expression in females than in males. In the endometrium, CYP3A4 and CYP3A43 are down-regulated by estrogen, whereas CYP3A5 is expressed at higher levels during the secretory phase. CYP3A7 was not detected in the endometrium. In addition, the CYP3A subfamily showed increased mRNA expression in the liver as age increased. The expression levels of total CYP3A protein and total CYP3A mRNA showed good correlation. Despite apparent regulation of CYP3A4 mRNA expression by estrogen, the effects of estrogen may be overshadowed by additional regulators of gene expression.

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.