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Accession: PRJNA128741 ID: 128741

A quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples (human)

See Genome Information for Homo sapiens
MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p<0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (3035), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p<0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). Our study aims to demonstrate the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies. Overall design: FFPE samples from 4 benign, reactive lymph nodes and 3 mantle cell lymphomas. Paired fresh-frozen and FFPE samples (3 benign reactive lymph nodes and 3 mantle cell lymphomas) were used for comparison. We also tested RNA samples at different concentrations (10, 25, 50, 100 and 200 ng/uL) as well as duplicate experiments for these RNA concentrations. Distinct RNA dilutions were also compared (5X, 7.5X, 30X and 62.5X dilution) at RNA concentrations of 66.7 ng/uL, 100 ng/uL and 200 ng/uL).
AccessionPRJNA128741; GEO: GSE22264
Data TypeTranscriptome or Gene expression
ScopeMultiisolate
OrganismHomo sapiens[Taxonomy ID: 9606]
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo; Homo sapiens
PublicationsGoswami RS et al., "Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples.", BMC Biotechnol, 2010 Jun 23;10:47
SubmissionRegistration date: 24-Jun-2010
Applied Molecular Oncology, University Health Network
RelevanceMedical
Project Data:
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PubMed1
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Other datasets
GEO DataSets1
GEO Data Details
ParameterValue
Data volume, Spots13056
Data volume, Processed Mbytes1
Data volume, Supplementary Mbytes1

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