Gene expression array testing of FFPE archival breast tumor samples: an optimized protocol for WG-DASL sample preparation.

Author(s): Ton CC, Vartanian N, Chai X, Lin MG, Yuan X, Malone KE, Li CI, Dawson A, Sather C, Delrow J, Hsu L, Porter PL

Publication: Breast Cancer Res Treat, 2011, Vol. 125, Page 879-83

PubMed ID: 20842525 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of RNA extraction method on successful analysis of FFPE breast tumors using the DASL assay. Paraffin blocks were stored for 11-17 years prior to extraction.

   Summary of Findings:

   When RNA was extracted using a modified Qiagen FFPE RNeasy kit protocol, including double the xylene deparaffinization steps, ethanol wash steps, and extension of proteinase k digestion to 2 h, approximately 8 fold more RNA was obtained than when RNA was extracted using the Roche Hi Pure kit. Further, the purity of the extracted RNA, as determined by spectrophotometer, was higher using the modified Qiagen protocol. Cycle threshold (CT) values for the RPL13a assay were above the recommended value of 29 for approximately 50% of specimens, but 100% of specimens extracted by the modified Qiagen protocol yielded DASL results, indicating the recommended CT value may not be a predictor of DASL success when extraction is with the modified Qiagen protocol. In contrast, when specimens were extracted using the Roche kit, only 8 of 10 specimens with a CT of less than 29 yielded DASL results. The number of detected probes in the DASL assay was similar to that previously reported in frozen lung tissue. Hierarchical clustering based on 729 probes properly grouped technical replicates in all cases and distinguished tumors based on ER status in >90% of specimens. A reexamination of the tumors misclassified as ER+ showed they had the lowest ER IHC scores of the cohort.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	DASL
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Roche Hi Pure FFPE RNA kit Modified Qiagen RNeasy FFPE kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	RPL13A
   Immunohistochemistry Specific	Targeted peptide/protein	Estrogen receptor

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