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


METHODS
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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the percentage of transcripts detected on the Affymetrix U133 2.0 human genome GeneChip.


RESULTS
The median RNA yield was 1.9 microg/mg tissue (papillary carcinoma 2.1 microg/mg, range 0.2-7.2 microg/mg; follicular carcinoma 2.4 microg/mg, range 0.2-3.2 microg/mg; goitre 1.4 microg/mg, range 0.1-5.4 microg/mg; follicular adenoma 1.6 microg/mg, range 0.1-6.2 microg/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%).


CONCLUSIONS
Age and entity independent RNA suitable for expression profiling can be extracted from long-term stored fresh-frozen human thyroid tissues.
Walter
Martin A
MA
Institute of Nuclear Medicine, University Hospital Basel, Switzerland. m.a.walter@gmx.net
Seboek
Dalma
D
Demougin
Philippe
P
Bubendorf
Lukas
L
Oberholzer
Martin
M
Müller-Brand
Jan
J
Müller
Beat
B
D018263:Adenocarcinoma, Follicular / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D000236:Adenoma / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D002291:Carcinoma, Papillary / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006042:Goiter / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D009926:Organ Preservation; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D013961:Thyroid Gland; D013964:Thyroid Neoplasms / Q000737:chemistry / Q000235:genetics*; D013997:Time Factors
D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases