Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

Background: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.

Results: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).

Conclusion: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

An in vitro investigation of endocrine disrupting effects of tricothecenes deoxynivalenol (DON), T-2, HT-2 toxins

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

Multiplex RT-PCR for the detection of leukemia-associated translocations - Validation and application to routine molecular diagnostic practice

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [He-mavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARalpha/t (15; 17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 x 10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 x 10(2) to 2 x 10(8) copies/mu l. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease. (C) 2010 Elsevier B.V. All rights reserved.

Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone

Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated.

Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR-based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB.

Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis.

Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV.

Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR-based techniques.

Novel hydrolysis-probe based qPCR assay to detect saxitoxin transcripts of dinoflagellates in environmental samples

Paralytic Shellfish Poisoning (PSP) is a serious human illness caused by ingestion of seafood enriched with paralytic shellfish toxins (PSTs). PSTs are neurotoxic compounds produced by marine dinoflagellates, specifically by Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. Every year, massive monitoring of PSTs and their producers is undertaken worldwide to avoid PSP incidences. Here we developed a sensitive, hydrolysis probe-based quantitative PCR (qPCR) assay to detect a gene essential for PST synthesis across different dinoflagellate species and genera and tested it on cDNA generated from environmental samples spiked with Alexandrium minutum or Alexandrium fundyense cells. The assay was then applied to two environmental sample series from Norway and Spain and the results were complemented with cell counts, LSU-based microarray data and toxin measurements (enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor method). The overall agreement between the results of the qPCR assay and the complementary data was good. The assay reliably detected sxtA transcripts from Alexandrium spp. and G. catenatum, even though Alexandrium spp. cell concentrations were mostly so low that they could not be quantified microscopically. Agreement between the novel assay and toxin measurements or cell counts was generally good; the few inconsistencies observed were most likely due to disparate residence times of sxtA transcripts and PSTs in seawater, or, in the case of cell counts, to dissimilar sxtA4 transcript numbers per cell in different dinoflagellate strains or species. © 2013 Elsevier B.V.

qPCR utilization for the study of RNA Polymerase I transcription

Ribosome biogenesis is a fundamental cellular process tightly linked to cell growth and proliferation, which requires the coordinated transcription of all three nuclear polymerases. Synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I) has been suggested as a key regulator of ribosome biogenesis, and there is a strong link between transcription of ribosomal RNAs and cellular proliferation. This makes Pol I transcription a valid and attractive target for anticancer therapy. At the moment however there are only a small number of compounds that act as specific inhibitors of Pol I transcription and this makes it very difficult for the development of drugs which would target rRNA transcription and consequently ribosome biogenesis. Therefore, to aid in the development of new inhibitors of Pol I, high-throughput methods to monitor and detect changes in Pol I activity need to be developed. This current study aimed to address the question of whether or not quantitative PCR (qPCR) could be used to detect changes in rRNA production in cells under different conditions that repress Pol I activity i.e. serum starvation and drug treatment. Our results have shown that using primers and a hydrolysis probe designed for the 5’ETS region of the pre-rRNA molecule, rRNA levels in both treated and untreated cells could be determined by using qPCR.
Amplification resulted in formation of a single product and S1 nuclease protection assay confirmed the down-regulation of Pol I transcription. Following serum-starvation and drug treatment there was a dramatic reduction in the amount of 5’ETS transcript quantitated by both Sybr Green chemistry and the use of a fluorescently labelled hydrolysis probe. The optimization of the qPCR strategy will be discussed.