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

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Abstract
Isolation of high-quality RNA from fresh-frozen thyroid tissues stored for more than a decade would open novel options for gene expression profiling. Herein, we describe successful extraction of high-integrity RNA from human thyroid tissues that were stored for more than a decade. Seventy-nine samples (15 goitres, 20 follicular adenomas, 30 papillary carcinomas, 14 follicular carcinomas) that were shock-frozen in isopentane and stored for a median of 11 years (range 1-16 years) were processed using standard precipitation and column filtration techniques. RNA integrity was assessed by electrophoresis using the RNA integrity number (RIN) algorithm and by gene expression profiling determining the 3’/5’ ratio of the glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) gene and the percentage of transcripts detected on the Affymetrix U133 2.0 human genome GeneChip. The median RNA yield was 1.9 μg/mg tissue (papillary carcinoma 2.1 μg/mg, range 0.2-7.2 μg/mg; follicular carcinoma 2.4 mg/mg, range 0.2-3.2 μg/mg; goitre 1.4 μg/mg, range 0.1-5.4 μg/mg; follicular adenoma 1.6 μg/mg, range 0.1-6.2 μg/mg; p = 0.46) with an 8.6 (7.3-9.8) median RIN. The median GAPDH gene 3’/5’ ratio was 1.43 (1.34-1.52) and the median percentage of present calls was 48.1% (42.7-52.0%). Age and entity independent RNA suitable for expression profiling can be extracted from long-term stored fresh-frozen human thyroid tissues.