From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues.
Author(s): Belder N, Coskun Ö, Doganay Erdogan B, Ilk O, Savas B, Ensari A, Özdağ H
Publication: Pathol Res Pract, 2016, Vol. 212(8), Page 678-85
PubMed ID: 27161306 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of deparaffinization method and RNA extraction method on RNA yield and purity and compared RNA amplification and labeling methods and microarray types on percentage of present calls using RNA from formalin-fixed paraffin-embedded (FFPE) specimens.
Conclusion of Paper
The highest RNA yields and purity were obtained using the modified deparaffinization procedure along with the RNeasy FFPE kit. The percent present calls were more than 2-fold higher when RNA was labeled and amplified using the Ovation FFPE WTA system rather than the 3’IVT kit and were slightly higher using the U133_X3P rather than the HG U133 Plus 2.0 array.
Studies
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Study Purpose
This study investigated the effects of deparaffinization method and RNA extraction kit on the yield, purity, and integrity of RNA from FFPE specimens. Six 10-year-old FFPE colorectal tumor specimens from four patients were sectioned at 8µm and tumor areas were determined by H&E staining. Four 8µm sections were deparaffinized by placing in stationary xylene for 10 min twice and washing in ethanol twice (modified procedure) or by vortexing in xylene, centrifugation, and washing once in ethanol (RNeasy FFPE method). Areas of deparrafinized specimens containing >90% tumor were macrodissected. RNA was extracted from specimens deparaffinized using the modified method using the RNeasy FFPE kit, Trizol (modified deparaffinization), and the PicoPure RNA isolation kits and from specimens deparaffinized using the RNeasy FFPE method with the RNEasy FFPE kit. RNA was amplified and labeled using the 3’IVT kit and labeled with the Ovation FFPE WTA system
Summary of Findings:
Deparaffinization using the modified procedure resulted in up to 3-fold higher RNA yields and increased OD 260/280 and OD 260/230 ratios compared to specimens deparaffinized using the RNeasy FFPE method. RNA yields and purity as determined by spectrophotometer were highest when extraction was with RNAeasy FFPE, next highest using Trizol, and lowest with PicoPure. Similarly, RIN was able to be determined for all four specimens extracted using RNeasy (RIN= 2.0-2.5), one specimen extracted using PicoPure (RIN=2.2), and none of the specimens extracted using Trizol.
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) Analyte Extraction and Purification Analyte isolation method RNeasy FFPE kit
Trizol
PicoPure RNA Isolation kit
Analyte Extraction and Purification Deparaffinization Modified method
RNeasy FFPE method
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Study Purpose
This study investigated the effects of RNA amplification and labeling procedure and array type on the percent of present calls using RNA from FFPE specimens. RNA was extracted from macrodissected areas of four 8µm sections of four FFPE colorectal tumor specimens and three matched non-tumor control specimens using a modified deparaffinization method and the RNeasy FFPE kit. RNA was amplified and labeled using the 3’IVT kit and labeled with the Ovation FFPE WTA system (random hexamer and Oligo dT) and hybridized to the two array types.
Summary of Findings:
The percentage of present calls were higher when RNA amplification and labeling was performed using the Ovation FFPE WTA system than the 3’IVT kit (41.81-55.52% versus 13.0-25.13%). Slightly higher percentage of present calls was found using the U133_X3P rather than the HG U133 Plus 2.0 array. In the three specimens with matched control tissue, 504 genes were found to be differentially expressed in tumor specimens and clustering analysis based on these transcripts correctly separated tumor from control specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) DNA microarray Specific Type of array Affymetrix Human Genome U133 Plus 2.0
Affymetrix U133_ X3P
DNA microarray Specific Template modification 3’IVT kit
Ovation FFPE WTA system