The Influence of Immunohistochemistry on mRNA Recovery from Microdissected Frozen and Formalin-Fixed, Paraffin-Embedded Sections
Author(s): Gjerdrum L, Abrahamsen HN, Villegas B, Sorensen BS, Schmidt H, Hamilton-Dutoit SJ
Publication: Diagn Mol Path, 2004, Vol. 13, Page 224
PubMed ID: 15538113 PubMed Review Paper? No
Purpose of Paper
This paper assessed the effects of slide storage and immunohistochemical staining on mRNA subsequently isolated from microdissected cells from formalin-fixed paraffin-embedded (FFPE) and OCT-embedded frozen tissue sections.
Conclusion of Paper
RNA extracted from immunohistochemically stained tissue sections yielded detectable although significantly reduced RNA and gene expression levels in comparison to hematoxylin stained controls for both FFPE and OCT embedded frozen tissue sections. Storage of tissue section slides at room temperature, 4 or -80 degrees C for up to 90 days did not significantly affect RNA quantity or gene expression levels.
Studies
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Study Purpose
To asses the effects of immunohistochemical staining on mRNA levels and gene expression in FFPE and OCT-embedded frozen microdissected cells. All OCT-embedded sections were post-fixed in acetone. Specimens underwent immunohistochemical or hematoxylin staining prior to microdissection.
Summary of Findings:
RNA quantity, the number of positive cases, and mean gene expression levels (beta-actin, beta2-microglobulin) were significantly reduced, as determined by real time quantitative RT-PCR analysis of immunohistochemically stained tissue compared to hematoxylin stained controls. While significant effects were observed with both frozen and FFPE tissues, the decrease in mean gene expression was more robust in FFPE tissue. Effects were observed for all immunohistochemical targets and genes measured.
Biospecimens
Preservative Types
- Formalin
- OCT
Diagnoses:
- Neoplastic
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Targeted nucleic acid beta-actin
beta 2-microglobulin
Real-time qRT-PCR Specific Type of tissue stain Immunohistochemistry
H&E
Biospecimen Preservation Type of fixation/preservation OCT
Formalin (buffered)
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Study Purpose
To determine the effect of FFPE slide storage (room temperature, 4 or -80 degrees C) on mRNA yield and gene expression.
Summary of Findings:
mRNA was quantifiable in all samples stored at room temperature for up to 90 days, but only in 2 of 9 samples after 180 days of storage at room temperature. Beta-actin mRNA levels were comparable among slides stored for 1 to 90 days, but were drastically reduced after 180 days of storage. Similar reductions in PCR success were observed when the sections were stored at 4 or -80 degrees C for 180 days compared to 90 days or less..
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 day
30 days
90 days
180 days
Storage Storage temperature Room temperature
4 degrees C
-80 degrees C
Real-time qRT-PCR Specific Targeted nucleic acid beta-actin
B2M