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Extraction of high-integrity RNA suitable for microarray gene expression analysis from long-term stored human thyroid tissues.

Author(s): Walter MA, Seboek D, Demougin P, Bubendorf L, Oberholzer M, Müller-Brand J, Müller B

Publication: Pathology, 2006, Vol. 38, Page 249-53

PubMed ID: 16753748 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if RNA integrity and expression in malignant and non-malignant thyroid tissue are adversely affected by prolonged storage (1-16 y) of snap-frozen specimens at -70°C.  In total, 79 surgically resected thyroid specimens that included malignant (30 papillary carcinomas, 14 follicular carcinomas) and non-malignant (15 goiters, 20 follicular adenomas) tissue were used for analysis.  Within 15-30 min of surgical resection, specimens were embedded in OCT compound, snap-frozen in isopentane pre-cooled with liquid nitrogen, and stored at -70°C for 1-16 y until analysis.  RNA was isolated from 10 mg of tissue by Trizol/chloroform extraction, purified with RNeasy mini-spin columns, and treated with DNAse.  RNA integrity number (RIN) was determined with a Bioanlyzer 2100.  Gene expression profiling and the 3’/5’ ratio of GAPDH transcripts were assessed using 5 µg of total RNA and a Human U133 Plus 2.0 Genechip.

   Summary of Findings:

   RNA yields from malignant and nonmalignant thyroid tissue specimens did not differ significantly, although higher median RNA yields were observed from malignant tissue (2.1-2.4 µg/mg) than non-malignant tissue (1.4-1.6 µg/mg).  Multiple regression analysis revealed that RIN was not correlated to tissue storage duration (r2=0.012, p=0.34).  RIN also did not differ significantly between RNA extracted from malignant and non-malignant thyroid tissue (p=0.10).  The percentage of genes represented on the Human U333 Plus 2.0 array that were expressed in goiter (51.5%), follicular adenoma (48.1%), papillary carcinoma (52.0%), and follicular carcinoma (42.7%) specimens were within the expected range (30-55%).  The 3’/5’ ratio of GAPDH transcripts was also well below the threshold for degradation (3.0) for all sample types: goiter (1.43), follicular adenoma (1.34), papillary carcinoma (1.52), and follicular carcinoma (1.29).  Collectively, the results led the authors to conclude that RNA isolated from snap-frozen malignant and non-malignant thyroid tissue that has been stored at -70°C for up to 11 y is of suitable quality for gene expression profiling using microarray.

   Biospecimens

   • Tissue - Thyroid Gland

   Preservative Types

   • OCT
   • Frozen

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Malignant Non-malignant
   Storage	Storage duration	1-16 y
   DNA microarray Specific	Targeted nucleic acid	GAPDH

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