ArticleTranslational oncology2014
The importance of reference gene analysis of formalin-fixed, paraffin-embedded samples from sarcoma patients - an often underestimated problem.
Article in Translational oncology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 14 citations in OpenAlex.
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- Characteristics and Spatially Defined Immune (micro)landscapes of Early-stage PD-L1-positive Triple-negative Breast Cancer.Clinical cancer research : an official journal of the American Association for Cancer Research · 2021Article
- Technical considerations when designing a gene expression panel for renal transplant diagnosis.Scientific reports · 2020Article
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- Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues.BMC medical genetics · 2019Article
- Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society · 2019Article
- Pan-Cancer Analysis of TCGA Data Revealed Promising Reference Genes for qPCR Normalization.Frontiers in genetics · 2019Article
- Platforms for Single-Cell Collection and Analysis.International journal of molecular sciences · 2018Review
- A prognostic profile of hypoxia-induced genes for localised high-grade soft tissue sarcoma.British journal of cancer · 2016Article
Corrections and comments
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
objectiveReverse transcription quantitative real-time polymerase chain reaction is efficient for quantification of gene expression, but the choice of reference genes is of paramount importance as it is essential for correct interpretation of data. This is complicated by the fact that the materials often available are routinely collected formalin-fixed, paraffin-embedded (FFPE) samples in which the mRNA is known to be highly degraded. The purpose of this study was to investigate 22 potential reference genes in sarcoma FFPE samples and to study the variation in expression level within different samples taken from the same tumor and between different histologic types.
methodsTwenty-nine patients treated for sarcoma were enrolled. The samples encompassed 82 (FFPE) specimens. Extraction of total RNA from 7-μm FFPE sections was performed using a fully automated, bead-base RNA isolation procedure, and 22 potential reference genes were analyzed by reverse transcription quantitative real-time polymerase chain reaction. The stability of the genes was analyzed by RealTime Statminer. The intrasamples variation and the interclass correlation coefficients were calculated. The linear regression model was used to calculate the degradation of the mRNA over time.
resultsThe quality of RNA was sufficient for analysis in 84% of the samples. Recommended reference genes differed with histologic types. However, PPIA, SF3A1, and MRPL19 were stably expressed regardless of the histologic type included. The variation in ∆Cq value for samples from the same patients was similar to the variation between patients. It was possible to compensate for the time-dependent degradation of the mRNA when normalization was made using the selected reference genes.
conclusionPPIA, SF3A1, and MRPL19 are suitable reference genes for normalization in gene expression studies of FFPE samples from sarcoma regardless of the histology.
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