Unknown,Transcriptomics,Genomics,Proteomics

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Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

ABSTRACT: INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues. Thirty samples from human cancers processed in different ways before RNA extraction: (i) Fresh-Freezing (FF), (ii) Standard Fixation with formalin (SF), (iii) Cold-Fixation (CF), a new formalin fixation procedure preserving nucleic acids. The first six samples compare CF with SF and FF in one colorectal cancer (CRC) specimen and one breast cancer (BRCa) specimen. The subsequent 24 samples compare only CF with FF in various cancer specimens. Expression data are neither normalized nor backound subtracted to preserve the original signal distribution for each sample, which is essential to compare expression profiles obtained after different tissue preservation methods.

ORGANISM(S): Homo sapiens

SUBMITTER: Enzo Medico

PROVIDER: E-GEOD-27175 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Formalin fixation at low temperature better preserves nucleic acid integrity.

Bussolati Gianni G Annaratone Laura L Medico Enzo E D'Armento Giuseppe G Sapino Anna A

PloS one 20110615 6

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in forma ...[more]

PMID: 21698245

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Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Dataset Information

Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Publications

Formalin fixation at low temperature better preserves nucleic acid integrity.

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