Impact of Fixative on Recovery of mRNA From Paraffin-Embedded Tissue

Abstract

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Differential gene expression profiles of neurothekeomas and nerve sheath myxomas by microarray analysis

“…Cumulatively, these lead to suboptimal nucleic acid quality and hampered reverse transcription and amplification reactions. [57][58][59][60] Thus, in the past, gene expression profile studies were restricted to tissues with higher intrinsic nucleic acid quality, such as cell lines, fresh blood or fresh frozen tissue. [61][62][63][64][65][66] Recent biotechnological advances have improved RNA isolation and amplification techniques, resulting in successful use of formalin-fixed paraffinembedded tissues to identify gene signatures in human tumors.…”

RNA expression analysis of formalin-fixed paraffin-embedded tumors

“…The variables that determine which blocks provide higher quality RNA are not clear from our analysis, but our data would be consistent with other work suggesting that the manner and details of formalin fixation are the crucial variables determining RNA quality from FFPE samples. 8,11,37,38 Given that degradation in FFPE samples is unpredictable and does not solely correlate with block age, we believe the significant contribution of this work is the ability to discern which RNA samples will provide useful microarray signatures before hybridization. The empirically determined criteria in this work account for both quantitative and qualitative problems with FFPE RNA as they address RNA degradation (by TaqMan) and inability to incorporate Cy-dye label (by Nanodrop).…”

“…RNA undergoes significant chemical modification by formalin and further degradation during storage. [8][9][10][11][12] Recently, however, there have been successful reports of the use of total RNA isolated from FFPE for RT-PCR assays. 3,[12][13][14][15][16] For example, Cronin et al 12 designed a 92-gene assay using RNA extracted from FFPE breast cancer specimens dating from 1985 to 2001, which yielded analyzable data in all tested specimens.…”

Gene expression analysis using long-term preserved formalin-fixed and paraffin-embedded tissue of non-small cell lung cancer

“…The main reason why FFPE derived RNA is not routinely used in gene expression studies is that the process of formalin fixation and paraffin embedding affects the RNA quality. The process is designed to preserve tissue morphology, but modifies RNA and degraded RNA molecules with limited Poly-A tail and cross linking artefacts are commonly noted (3)(4)(5)(6)(7).…”