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MRNA and miRNA expression profiles for fresh frozen periampullary adenocarcinomas (pancreatobiliary and intestinal type) and adjacent normal tissues.

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE60980 | GEO | 2015/11/06

SECONDARY ACCESSION(S): PRJNA260032

REPOSITORIES: GEO

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Project description:BACKGROUND:The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. RESULTS:In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. CONCLUSIONS:Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

Project description:Medical research projects become increasingly dependent on biobanked tissue of high quality because the reliability of gene expression is affected by the quality of extracted RNA. Hence, the present study aimed to determine if clinical, surgical, histological, and molecular parameters influence RNA quality of normal and tumoral frozen colonic tissues. RNA Quality Index (RQI) was evaluated on 241 adenocarcinomas and 115 matched normal frozen colon tissues collected between October 2006 and December 2012. RQI results were compared to patients' age and sex, tumor site, kind of surgery, anastomosis failure, adenocarcinoma type and grade, tumor cell percentage, necrosis extent, HIF-1? and cleaved caspase-3 immunohistochemistry, and BRAF, KRAS and microsatellites status. The RQI was significantly higher in colon cancer tissue than in matched normal tissue. RQI from left-sided colonic cancers was significantly higher than RQI from right-sided cancers. The RNA quality was not affected by ischemia and storage duration. According to histological control, 7.9% of the samples were unsatisfactory because of inadequate sampling. Biobanked tumoral tissues with RQI ?5 had lower malignant cells to stromal cells ratio than samples with RQI <5 (p <0.05). Cellularity, necrosis extent and mucinous component did not influence RQI results. Cleaved caspase-3 and HIF-1? immunolabelling were not correlated to RQI. BRAF, KRAS and microsatellites molecular status did not influence RNA quality. Multivariate analysis revealed that the tumor location, the surgical approach (laparoscopy versus open colectomy) and the occurrence of anastomotic leakage were the only parameters influencing significantly RQI results of tumor samples. We failed to identify parameter influencing RQI of normal colon samples. These data suggest that RNA quality of colonic adenocarcinoma biospecimens is determined by clinical and surgical parameters. More attention should be paid during the biobanking procedure of right-sided colon cancer or laparoscopic colectomy specimen. Histological quality control remains essential to control sampling accuracy.

Dataset Information

MRNA and miRNA expression profiles for fresh frozen periampullary adenocarcinomas (pancreatobiliary and intestinal type) and adjacent normal tissues.

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