An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis.
Author(s): Karsten SL, Van Deerlin VM, Sabatti C, Gill LH, Geschwind DH
Publication: Nucleic Acids Res, 2002, Vol. 30, Page E4
PubMed ID: 11788730 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
This study compared cDNA microarray performance of RNA extracted from snap frozen, formalin fixed, or ethanol fixed postmortem brain specimens, as well as evaluated the use of tyramide signal amplification. All brain specimens evaluated were collected at autopsy.
Summary of Findings:
A comparison of cDNA microarray results revealed that the percentage of spots generating a signal differed as a result of preservation method, with 94%, 99%, and 56% of spots generating a signal for RNA extracted from snap frozen, ethanol fixed, and formalin fixed specimens, respectively. Direct labeling of RNA extracted from formalin fixed specimens reduced the percentage of spots generating a signal to 30%. Formalin fixed specimens also generated low signal intensity and high background compared to the other preservation methods examined. Reproducibility was high for frozen specimens, acceptable for ethanol fixed specimens, and highly variable for formalin fixed specimens. cDNA synthesis and dye incorporation efficiency were also reduced in formalin fixed specimens, which were not improved by inclusion of a RNA preheating step at 70 degrees C for 1 hour.
Biospecimens
Preservative Types
- Formalin
- Frozen
- Ethanol
Diagnoses:
- Not specified
- Autopsy
Platform:
Analyte Technology Platform RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Ethanol
Formalin (buffered)
Snap frozen
DNA microarray Specific Signal amplification Tyramide signal amplification
No signal amplification
Preaquisition Postmortem interval 8-11 h
DNA microarray Specific RNA incubation temperature 70 degrees C for 1 h