Optimization of the method of RNA isolation from paraffin blocks to assess gene expression in breast cancer.
Author(s): Jarzab M, Rózanowski P, Kowalska M, Zebracka J, Rudnicka L, Stobiecka E, Jarzab B, Stachura J, Pawlega J
Publication: Pol J Pathol, 2008, Vol. 59, Page 85-91
PubMed ID: 18669173 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of section number, double deparaffinization, DNase treatment, and storage duration on RNA yield and integrity from FFPE breast carcinoma specimens. RNA was extracted from 6-7 um sections using the RNeasy FFPE kit.
Summary of Findings:
Extraction of RNA using DNase and the gDNA eliminator columns led to inadequate RNA yield while RNA extracted using only gDNA eliminator columns for DNA removal had 4-fold higher concentrations. Higher concentrations of RNA were obtained from 5 or 8 sections than from 3 sections (279, 302 and 178 ng/uL, respectively), but the difference was only significant when 8 sections were used instead of 3. Further, while there was no effect of double deparaffinization, RNA yield increased with lab personal experience. RNA yield was not affected by storage duration, but RNA from blocks stored for 1 week had weak but present 18S and 28S ribosomal RNA peaks that were not present in RNA from archival blocks.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 week
14-24 years
Analyte Extraction and Purification Nucleic acid digestion DNAse treatment and gDNA eliminator
gDNA eliminator column
Biospecimen Aliquots and Components Aliquot size/volume 3 sections
5 sections
8 sections
Analyte Extraction and Purification Deparaffinization Single deparaffinization in xylene
Double deparaffinization in xylene