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

Author(s): Strand C, Enell J, Hedenfalk I, Fernö M

Publication: BMC Mol Biol, 2007, Vol. 8, Page 38

PubMed ID: 17519006 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare different RNA quality assessment methods and to determine if RNA degradation affects relative levels of specific transcripts. Three different methods of RNA quality assessment using bioanalyzer data was examined: visual inspection, which was based upon the presence of two distinct ribosomal RNA peaks and a flat baseline; the ratio method, which was based on a threshold of 0.65; and RNA integrity number (RIN), which was based on a threshold of 6. Breast tumors collected from six patients were frozen in liquid nitrogen and stored at -80 degrees C before analysis. Frozen specimens were divided and case-matched aliquots were thawed at room temperature in a timecourse study to introduce RNA degradation.

   Summary of Findings:

   For the majority of specimens, RNA degradation was apparent after a thaw duration of 10 min, although 2 of the 6 specimens were affected after 2-3 min at room temperature, and 1 remained unaffected after 30 min. When evaluation methods and respective quality thresholds were compared by pair-wise comparisons, 83% (20/24) of specimens displayed concordant results for all three methods. Concordance for RNA quality determination was highest (96%; 23/34) between visual inspection and RIN methods. Unsupervised hierarchical clustering analysis of gene expression profiles determined by microarray from samples collected from the same tumor but subjected to different cold ischemia times produced two main groups: specimens with good or partly degraded RNA, and those with degraded RNA. This pattern of clustering was observed for each evaluation method and the respective quality thresholds imparted. However, the number of samples that did not cluster based upon the RNA quality pattern described above did differ among assessment methods, as visual inspection of bioanalyzer data yielded 1 sample that clustered outside of the RNA quality pattern while the ratio method yielded 5 samples. When microarray data was assigned into two groups based upon RIN threshold (RIN <6, RIN ≥6), 9 of the 10 genes that were differentially expressed between the two groups were present at lower levels in samples with a RIN <6.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Thaw duration	50 s 2-3 min 10 min 30 min
   Automated electrophoresis/Bioanalyzer Specific	Quality metrics	Visual inspection Ratio method RIN

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