RNA expression analysis of formalin-fixed paraffin-embedded tumors.
Author(s): Penland SK, Keku TO, Torrice C, He X, Krishnamurthy J, Hoadley KA, Woosley JT, Thomas NE, Perou CM, Sandler RS, Sharpless NE
Publication: Lab Invest, 2007, Vol. 87, Page 383-91
PubMed ID: 17297435 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to assess the quality and quantity of RNA extracted from 2- to 8-year-old FFPE tissue blocks.
Summary of Findings:
While sufficient RNA for TaqMan-based real time quantitative RT-PCR (qRT-PCR) analysis was obtained for all FFPE specimens, RNA yields were variable and not correlated to the age of the paraffin block or the size of the extracted specimen. Determination of RNA quality by OD 260/280 ratio, gel electrophoresis, or bioanalyzer was unsuccessful due to large sample requirements or failure to discern the degree of degradation. RNA integrity, as measured by 3'/5' ratio, was only successfully determined for archived FFPE specimens when a TaqMan strategy was employed, as a bioanalyzer and SYBR-based real time qRT-PCR lacked specificity. Of note, 3' and 5' primers were located 300 bp apart in the 3' end of the beta-actin transcript.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Melanoma
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 2-8 yr
Real-time qRT-PCR Specific Targeted nucleic acid Beta-actin
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Study Purpose
The purpose of this paper was to develop RNA quality criteria and thresholds that would identify archival formalin-fixed, paraffin-embedded (FFPE) tumor specimens that would result in informative microarray analysis.
Summary of Findings:
The authors identified several key RNA yield and quality thresholds predictive of successful microarray analysis using archived FFPE specimens. FFPE extracted RNA successfully applied for microarray analysis (1) had a yield >600 ng total RNA (as determined by a multi-wavelength spectrophotometer); (2) an OD 260/280 ratio >1.5; (3) a 3'/5' ratio <100 (as determined by TaqMan-based real time qRT-PCR); and (4) Cy-dye incorporation >4.5 pmol/ng. When the above criteria were applied as a screen, 20 FFPE specimens of 157 were identified with a 100% microarray success rate (20/20). When no screen was applied the microarray success rate for FFPE specimens was 24% with 37 of 157 FFPE specimens producing informative microarray results.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Melanoma
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Spectrophotometry Specific Quality metrics RNA yield
OD 260/280 ratio
3'/5' ratio
Cy-dye incorporation
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Study Purpose
The purpose of this study was to compare the microarray performance of RNA extracted from FFPE tissue and case-matched fresh-frozen tissue.
Summary of Findings:
One hundred percent of the samples derived from frozen specimens resulted in informative microarray analysis, while just 50% of FFPE case-matched specimens were successful. While the rate of success was increased by application of newly devised RNA quantity and quality selection criteria, the authors fail to report this value. Of the FFPE specimens that yielded informative arrays, the quality of expression data was comparable to results obtained from frozen specimens.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Melanoma
Platform:
Analyte Technology Platform RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen