Surgical Specimens of Colorectal Cancer Fixed with PAXgene Tissue System Preserve High-Quality RNA.
Author(s): Hara K, Watanabe A, Matsumoto S, Matsuda Y, Kuwata T, Kan H, Yamada T, Koizumi M, Shinji S, Yamagishi A, Ishiwata T, Naito Z, Shimada T, Uchida E
Publication: Biopreserv Biobank, 2015, Vol. 13, Page 325-34
PubMed ID: 26484572 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of preservation method and fixation time on RNA yield, integrity, and stability; tissue morphology; and immunohistochemical analysis of colorectal tumor tissue.
Conclusion of Paper
RNA yield did not differ among specimens preserved with RNAlater, PAXgene, and formalin. However, mean RNA integrity numbers (RINs) were significantly lower in specimens fixed in PAXgene (6.5) and buffered or nonbuffered formalin for 6 or 48 h (1.8-2.3) compared to RNAlater-preserved specimens (8.7)(P<0.01). Transcript stability of GAPDH, GPX1, VDAC2, ABL1, EGR1, assessed by the cycle threshold (Ct) value of amplicons approximately 100 and 500 bp in length was comparable among specimens preserved in RNAlater and PAXgene. While Ct values were significantly higher for FFPE specimens (regardless of fixative content or time in formalin) when compared to those preserved in RNAlater, Ct values were higher for all transcripts (with the exception of GAPDH) among FFPE specimens fixed in 48 h compared to those fixed for 6 h (P<0.05). Transcript expression (normalized to GAPDH) did not differ significantly between RNAlater and PAXgene preserved specimens for GPX1, VDAC2 or EGR1, although normalized ABL1 expression was significantly lower in PAXgene compared to RNAlater-preserved specimens (P<0.01). Among FFPE specimens, VDAC2 was the only transcript that did not display significantly lower mean normalized expression when between specimens fixed in buffered formalin were compared to those preserved in RNAlater. Limited morphological changes were observed among specimens fixed in PAXgene compared to formalin, but they did not influence tumor diagnosis. Immunohistochemical (IHC)-staining of Ki-67, CEA, and EGFR was comparable between PAXgene specimens and 6 h formalin-fixed specimens.
Studies
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Study Purpose
This study investigated the effects of preservation method and fixation time on RNA yield, integrity, and transcript stability as well as tissue morphology and immunohistochemical analysis of colorectal tumor tissue. Specimens from 10 colorectal cancer patients undergoing colectomy from October 2012 to January 2013 were divided into eight smaller pieces: one was placed in RNAlater and frozen at -80°C for 48–72 h; one was fixed in PAXgene Tissue FIX Solution for 2–4 h, transferred to PAXgene Tissue STABILIZER and stored at -20°C for 48 h followed by dehydration by ethanol, chloroform treatment, and then embedded in paraffin; and six pieces were fixed in non-buffered 20% formalin or 10% or 20% neutral-buffered formalin (NBF) for 6 or 48 h. RNA was extracted from RNAlater specimens using the RNeasy Mini Kit, from PAXgene specimens using the PAXgene Tissue RNA Kit and from formalin specimens using the RNeasy FFPE Kit. Total RNA yield was determined by spectrophotometer, and RNA integrity number (RIN) was determined by bioanalyzer. Real-time RT-PCR of GAPDH, GPX1, VDAC2, ABL1, and EGR1 short (95 bp, 133 bp, 115 bp, 130 bp, and 114 bp; respectively) and long amplicons (430 bp, 345 bp 580 bp, 467 bp, and 355 bp, respectively) was performed to evaluate transcript stability and degradation. PAXgene and formalin specimens (3 µm sections) were H&E-stained for morphology assessment or IHC-stained for Ki-67, CEA, and EGFR.
Summary of Findings:
RNA yield did not differ among specimens preserved with RNAlater, PAXgene, and formalin. However, mean RNA integrity numbers (RINs) were significantly lower in specimens fixed in PAXgene (6.5) and buffered or nonbuffered formalin for 6 or 48 h (1.8-2.3) compared to RNAlater-preserved specimens (8.7)(P<0.01). The percentage of specimens that yielded RNA with a RIN >7.0 was 90% for frozen specimens, 80% for PAXgene specimens, and 0% for formalin specimens. Transcript stability of GAPDH, GPX1, VDAC2, ABL1, EGR1, assessed by the cycle threshold (Ct) value of amplicons approximately 100 and 500 bp in length was comparable among specimens preserved in RNAlater and PAXgene. While Ct values were significantly higher for FFPE specimens (regardless of fixative content or time in formalin) when compared to those preserved in RNAlater, Ct values were higher for all transcripts (with the exception of GAPDH) among FFPE specimens fixed in 48 h compared to those fixed for 6 h (P<0.05). Transcript expression (normalized to GAPDH) did not differ significantly between RNAlater and PAXgene preserved specimens for GPX1, VDAC2 or EGR1, although normalized ABL1 expression was significantly lower in PAXgene compared to RNAlater-preserved specimens (P<0.01). Among FFPE specimens, VDAC2 was the only transcript that did not display significantly lower mean normalized expression when between specimens fixed in buffered formalin were compared to those preserved in RNAlater. Limited morphological changes were observed among specimens fixed in PAXgene compared to formalin, but they did not influence tumor diagnosis. Immunohistochemical (IHC)-staining of Ki-67, CEA, and EGFR was comparable between PAXgene specimens and 6 h formalin-fixed specimens.
Biospecimens
Preservative Types
- Formalin
- PAXgene
- RNAlater
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Morphology H-and-E microscopy RNA Automated electrophoresis/Bioanalyzer Protein Immunohistochemistry RNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Concentration of fixative 20% formalin
10% NBF
20% NBF
Biospecimen Preservation Time in fixative 6 h
48 h
Biospecimen Preservation Type of fixation/preservation RNAlater
PAXgene
Neutral buffered formalin with saline
Formalin (unbuffered)
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
GPX
VDAC2
ABL1
EGR1
Real-time qRT-PCR Specific Length of gene fragment 95-133 bp
345-580 bp
Immunohistochemistry Specific Targeted peptide/protein Ki-67
CEA
EGFR
Analyte Extraction and Purification Analyte isolation method RNeasy Mini Kit
PAXgene Tissue RNA Kit
RNeasy FFPE Kit