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Evaluation of small sample cDNA amplification for microdissected airway expression profiling in COPD
John V Gosselink1, Shizu Hayashi, Edmond Chau
1St. Paul's Hospital, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research and University of British Columbia, Vancouver, British Columbia. jgosselink@mrl.ubc.ca
COPD
|May 29, 2007
Summary
SMART amplification enables gene expression analysis in small airways of chronic obstructive pulmonary disease (COPD) patients. This method yields sufficient RNA quantity and quality for large-scale gene studies from laser-captured tissues.
Area of Science:
- Pulmonary Medicine
- Molecular Biology
- Genomics
Background:
- Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation due to small airway obstruction and emphysema.
- Laser capture microdissection (LCM) isolates specific tissues, but limited RNA quantity restricts gene expression studies in small airways.
- Evaluating gene expression in small airways is crucial for understanding COPD pathogenesis.
Purpose of the Study:
- To assess the efficacy of Clontech SMART amplification for gene expression analysis in small airways obtained via LCM.
- To determine if amplified RNA from microdissected airways is sufficient for large-scale gene expression profiling.
- To validate the accuracy of gene expression patterns after SMART amplification compared to unamplified RNA.
Main Methods:
- SMART amplification of commercial reference RNA and RNA from microdissected small airways.
- Gene expression analysis of amplified and unamplified RNA using quantitative real-time PCR.
- Correlation analysis (R-value) to compare gene expression levels between amplified and unamplified RNA samples.
- Linear regression analysis of delta Ct values for RNA preparations.
Main Results:
- SMART amplification of commercial RNA showed a high correlation (R=0.84) for 51 genes compared to unamplified RNA.
- Linear regression of delta Ct values yielded high correlation coefficients (R=0.95) for different RNA pairs, including COPD patient samples.
- Gene expression patterns in amplified RNA from microdissected small airways closely mirrored those of unamplified RNA across all COPD severity GOLD stages.
Conclusions:
- SMART amplification provides sufficient cDNA quantity and quality for comprehensive gene expression studies in laser-captured small airways.
- The amplification method effectively preserves the relative gene expression profiles present in the original unamplified RNA.
- This technique facilitates large-scale gene expression analysis in limited tissue samples, advancing COPD research.
