Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

help |

Analytical performance of a qRT-PCR assay to detect guanylyl cyclase C in FFPE lymph nodes of patients with colon cancer.

Author(s): Beaulieu M, Desaulniers M, Bertrand N, Deschesnes RG, Beaudry G, Garon G, Haince JF, Houde M, Holzer TJ

Publication: Diagn Mol Pathol, 2010, Vol. 19, Page 20-7

PubMed ID: 20186008 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of specimen storage duration and temperature, fat clearing agent, fixative type and duration, specimen staining and amplicon size on real-time PCR amplification of Guanylyl cyclase C (GCC) mRNA in formalin-fixed paraffin embedded (FFPE) colon and associated lymph nodes.

Conclusion of Paper

Prior to fixation, specimens were stored for 24 h at RT or up to 6 days at 4 degrees C without a change in GCC quantification by real-time PCR. Fixation time did not impact GCC quantification, but fixatives other than 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) resulted in decreased GCC amplification. No effect of fat clearing agent or tissue staining on amplification of GCC was observed. FFPE specimens were comparable to fresh in real-time PCR amplification of a 69 bp fragment of B-actin but longer fragments had higher CT values which increased further with specimen age. The authors conclude that real-time PCR can be used to accurately quantify GCC in paraffin embedded specimens, but that fixative type, storage duration and amplicon size can alter results.

Studies

Study Purpose

The purpose of this study was to determine the effects of storage of FFPE lymph nodes (1 month-12 years) and colon (fresh-1d) on RNA size distribution by gel electrophoresis and amplification of B-actin by real-time RT-PCR.

Summary of Findings:

Storage of FFPE colon for 1 day resulted in degradation of RNA fragments greater than 1000 bp when compared to fresh frozen specimens. Similarly FFPE lymph nodes stored between 1 month and 1 year had loss of RNA greater than 1000 bp. In specimens archived between 1 and 12 years, further degradation was apparent with most specimens predominantly consisting of RNA between 50-300 bp. As expected based on the gel electrophoresis results, all FFPE specimens showed increasing cycle threshold (CT) numbers with larger amplicons. However, the magnitude of this change was greater in specimens archived for 10 years (approximately 15 CT's) then in specimens archived for 2-3 months (approximately 6 CTs). In contrast, CT values from fresh frozen specimens only increased by 2 CTs over the amplicon size range studied.

Biospecimens

Preservative Types

Formalin
Frozen

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Normal Adjacent
Other diagnoses

Platform:

Analyte	Technology Platform
RNA	Real-time qRT-PCR
RNA	Electrophoresis

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Storage	Storage duration	1 d 1 month 2 months 3 months 4 months 5 months 6 months 1 year 2 years 3 years 5 years 8 years 9 years 11 years 12 years
Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
Biospecimen Acquisition	Biospecimen location	Colon lymph node Colon
Real-time qRT-PCR Specific	Length of gene fragment	69 bp 80 bp 98 bp 149 bp 167 bp 223 bp 241 bp

Study Purpose

The purpose of this study was to examine the effects of cold ischemia time at room temperature (6h to 7 d) or 4 degrees C (0 h to 7 d), fat clearing solution and prior specimen staining on amplification of GCC RNA by real-time RT-PCR.

Summary of Findings:

Cold ischemia times of up to 24 h at room temperature or 6 days at 4 degrees C did not affect amplification of GCC. Storage for 7 days at either room temperature or 4 degrees C resulted in 1% or fewer of the GCC copies being detected (p<0.01). No effect of tissue staining or fat clearing solution on GCC copy number determination was found.

Biospecimens

Tissue - Colorectal

Preservative Types

Formalin

Diagnoses:

Neoplastic - Normal Adjacent

Platform:

Analyte	Technology Platform
RNA	Real-time qRT-PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Acquisition	Pathology ink	Unstained Tissue marking dye
Analyte Extraction and Purification	Fat clearing	NBF, 95% ETOH, 100% EtOH, methyl salicylate Acetone, methyl salicylate 99% EtOH, xylene Lymph node revealing solution Glacial acetic acid-ethanol-distilled water-formaldehyde
Biospecimen Acquisition	Cold ischemia time	6 h 24 h 6 d 7 d

Study Purpose

The purpose of this study was to determine the effects of fixative type and duration on GCC copy number in a single colon specimen by real-time PCR.

Summary of Findings:

Fixation for 1 or 2 days with either 10% NBF or 4% PFA gave similar results. All other fixatives resulted in a reduced GCC copy number. Fixing of the specimens in Bouin's solution after fixing in 10% NBF also resulted in a decline in GCC copy number. Specimens fixed with ethanol (70% or 100%), 10% non-buffered formalin and Tissue fix performed particularly poorly allowing for quantification of less than 0.1% of GCC copies.

Biospecimens

Tissue - Colorectal

Preservative Types

Formalin
Other Preservative
Ethanol

Diagnoses:

Neoplastic - Normal Adjacent

Platform:

Analyte	Technology Platform
RNA	Real-time qRT-PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Time in fixative	1 d 2 d 1 h 2 h
Biospecimen Preservation	Type of fixation/preservation	B-5 fixative Bouin's fixative Carnoy's solution Ethanol Formalin (buffered) Formalin (unbuffered) Formol saline Methacarn Modified Davidson's fluid Tissue fix

You Recently Viewed

News and Announcements

Most Downloaded SOPs in 2024

New Articles on the GTEx Project are Now FREELY Available!

Just Published!

More...