Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue

Abstract

S U M M A R Y Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphatebuffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:1033-1042, 2008

A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

“…11,24,25 Alternatively, immunostaining of On the basis of the principle of heat-induced antigenretrieval techniques, we previously demonstrated that the use of antigen-retrieval buffer (pH 10) containing 1% SDS combined with high temperature resulted in excellent protein-extraction yield from archival FFPE tissue sections, without changing the antigenicity. 19 Therefore, we used antigen-retrieval buffer (Tris-HCl, pH 10) and Tris-based buffer containing detergent (Tris/Tricine/SDS, pH 8.2) as test bleaching buffers in addition to PBS as a control to investigate their effect on melanin-bleaching efficiency and downstream applications. All three alkaline diluents for H 2 O 2 showed good quality of histomorphology ( Figure 2).…”

“…[18][19][20] The specimens were rinsed briefly once in 100% ethanol, and then resuspended and ground in a solution of 4 M guanidine isothiocyanate, 20 mM sodium acetate, and 25 mM β-mercaptoethanol (pH 5.5) followed by 72 h incubation at 65°C with mild shaking. After incubation, RNA was isolated by phenol/chloroform extraction.…”

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…These are predominantly available as formalin-fixed paraffin-embedded (FFPE) tissues, which are suitable for IHC analysis, but due to the degradation and cross-linking present a technical challenge for nucleic acid analysis. 24 Several commercially available RNA assays based on detection of full-length E6/E7 transcripts, 25,26 as well as ''in house'' RNA assays based on detection of spliced E6*I/E7 transcripts, 15,16,27 have been described. These assays aim to amplify rather long cDNA sequence from cervical smear samples (up to 620 bp for full-length and up to 280 bp for spliced transcripts, respectively) and therefore are not suited for FFPE tissue analysis.…”

Diagnosis of Influenza from Respiratory Autopsy Tissues

“…Studies examining tissue fixation times and processing after collection suggest that fixation should commence as soon as possible but proceed no longer than 48 hours after collection. 46,47 With these data in mind, standardized protocols for efficient and effective tissue fixation and processing should be made available to pathologists and other public health professionals worldwide so as to provide high-quality tissue specimens that are ideal for use in both IHC and rRT-PCR assays.…”