RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Abstract

Background: Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.

Association of low tumor RNA integrity with response to chemotherapy in breast cancer patients

“…One post-treatment biopsy was not available for RIN assessment due to the lack of a visible lesion. Similar to previously reported findings [28][29][30], we observed that the RIN is a reliable measure of RNA integrity, as visualized by inspection of electropherograms of biopsy RNA preparations after capillary electrophoresis. The RIN value for a particular tumor RNA preparation was clearly proportional to the intensity of the 28S and 18S rRNA bands on electropherograms (Fig.…”

“…In the latter case, a RIN of 10 represents high quality, intact RNA, while 0 represents completely hydrolyzed RNA. The RIN has been shown to be superior to the 28S/18S rRNA ratio for reliably measuring RNA quality [28][29][30] and is emerging as the ''gold standard'' for RNA quality assessment in mammalian cell lines and tissues [31], including breast tumors [30].…”

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

“…Integrity of obtained RNA was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples had an RNA Integrity Number (RIN) greater than 9.4 [26]. Complementary RNA was prepared from 1 lg total RNA, using one-cycle target labeling and a control reagents kit (Affymetrix, Santa Clara, CA).…”

Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 Bioanalyzer™ capillary electrophoresis system

“…This is in line with a study by Copois et al [10] who compared different methods of RNA assessment-degradometer software [11], 28S to 18S ratio, and an in-house quality scale-with array quality assessed by determination of the 3′ to 5′ ratio of GAPDH and a clustering analysis of full array expression ("dispersion tree"). A similar conclusion was drawn from a small study comparing RNA quality of 24 frozen breast cancer samples assessed by RIN, visual inspection of the capillary electrophoretic trace, and the 28S to 18S ratio [12]. Jahn et al assessed bacterial RNA quality by RIN and found it to be critical for obtaining meaningful gene expression data.…”