A multiplex PCR MSI assay for endoscopic and liquid biopsy
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ABSTRACT:
PROVIDER: PRJEB52735 | ENA |
REPOSITORIES: ENA
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Project description:Accurate detection of constitutional mismatch repair deficiency by a simple, sequencing-based microsatellite instability assay
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Project description:MSI analysis using blood-derived MSI markers
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Project description:BackgroundPSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence.ResultsAn initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP). The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE) samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity.ConclusionsThe developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.
| S-EPMC3431995 | biostudies-literature
Project description:Human tissue kallikreins (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished only by enzyme-linked immunosorbent assays (ELISA). In this work, we developed a multiplex selected reaction monitoring (SRM) assay for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and then multiplex in a single assay. Assay performance was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in seminal plasma, sweat and cervico-vaginal fluid, with limits of detection ranging from 5 to 500 ng/mL. Analytical performance of the SRM assay was evaluated by measuring endogenous KLKs in relevant clinical samples and results were compared to selected ELISAs. The multiplex SRM assay was proven to be an accurate, reproducible, sensitive and highly specific alternative to the existing antibody-based assays. Finally, we used immunoenrichment-SRM and ELISA to unambiguously detect and quantify seminal plasma levels of kallikrein-4, a highly prostate-specific protein and a potential biomarker of prostate-related diseases. The presented multiplex SRM assay is an alternative analytical tool to study the biological and pathological roles of human KLKs.
2016-07-08 | PXD003324 | Pride
Project description:Comparison of two different multiplex PCR primer pools in amplifying target HPV types from plasmid templates. Template concentrations are either 1 ng or 1 pg. Templates are detected by type-spcific LDR probes hybridized on microarray.
2011-05-01 | E-MEXP-3154 | biostudies-arrayexpress
Project description:Comparison of two different multiplex PCR primer pools in amplifying target HPV types from plasmid templates. Template concentrations are either 10 pg or 100 pg. Templates are detected by type-spcific LDR probes hybridized on microarray.
2011-08-20 | E-MEXP-3268 | biostudies-arrayexpress
Project description:Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.
| S-EPMC3839403 | biostudies-other
Project description:Multiplex amplification of human sequences using SMART technology
2008-07-26 | E-MEXP-1638 | biostudies-arrayexpress