Effects of processing delay, formalin fixation, and immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sections.
Author(s): van Maldegem F, de Wit M, Morsink F, Musler A, Weegenaar J, van Noesel CJ
Publication: Diagn Mol Pathol, 2008, Vol. 17, Page 51-8
PubMed ID: 18303406 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of a prefixation delay (0 to 48 h) at different temperatures (room temperature, 4 degrees C) on RNA quality and RT-PCR success of three housekeeping genes (hypoxanthine phosphoribosyltransferase, porphobilinogen deaminase, and beta-2-microglobulin) using normal liver and stomach and ovarian carcinoma specimens.
Summary of Findings:
The authors report a progressive deterioration in RNA quality with increasing time between tissue excision and freezing. This was only prevented by storage at 4 degrees C compared to room temperature after a delay of 12 h or longer. Degradation was more rapid in liver than ovary, which showed no discernable degradation even up to 18 hours at room temperature. Amplicons of appropriate sizes (157-753 bp) were obtained for all samples regardless of storage conditions with the exception of the specimen stored at room temperature for 48 h, which did not yield a PCR product longer than 331 bp.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 h
2 h
4 h
8 h
12 h
24 h
48 h
Storage Storage temperature 4 degrees C
Room temperature
Biospecimen Acquisition Biospecimen location Liver
Ovary
RT-PCR Specific Length of gene fragment 150 bp
225 bp
350 bp
500 bp
750 bp
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Study Purpose
The purpose of this study was to determine the optimum fixation period for successful RT-PCR analysis of RNA isolated from FFPE tissue sections.
Summary of Findings:
RNA isolated from all FFPE tissue sections lacked distinct ribosomal bands regardless of fixation duration. RT-PCR success rate indicated that a fixation duration of 8 to 16 h was optimal, producing maximal length amplicons. Heat denaturation of RNA samples at 70 degrees C for 2 h prior to reverse transcription yielded the best RNA-PCR results for all tissues types analyzed (liver, ovary, stomach).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 2 h
4 h
8 h
16 h
24 h
48 h
RT-PCR Specific RNA incubation temperature 65 degrees C
70 degrees C
80 degrees C
RT-PCR Specific Incubation time 10 min
2 h
6 h
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Study Purpose
The purpose of this study was to optimize immunohistochemical staining (for vimentin) using FFPE specimens to permit subsequent RNA isolation and RT-PCR analysis of stained tissue sections.
Summary of Findings:
The authors report successful RT-PCR amplification of RNA subsequent to immunohistochemistry when use of sera is avoided (slide mounting, blocking agents, antibody diluents) and detection using the 3-amino-9-ethylcarbazole (AEC) method is employed.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Immunohistochemistry RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Immunohistochemistry Specific Reaction solution Normal goat serum
Bovine serum albumin
Serum-free protein block
No blocking solution
Immunohistochemistry Specific Antibody dilution/concentration Antibody diluted in phosphate buffered saline
Antibody diluted in normal antibody diluent
Immunohistochemistry Specific Detection method Horseradish peroxidase
3,3 diaminobenzidine (DAB)
3-amino-9-ethylcarbazole (AEC)
Fuschine acid
Fast-Red
Fast-Blue