Evaluation of small sample cDNA amplification for microdissected airway expression profiling in COPD.
Small airway obstruction and emphysematous destruction account for the airflow limitation that defines chronic obstructive pulmonary disease (COPD). While laser capture microdissection (LCM) allows gene expression studies in small airways separately from the surrounding parenchyma, tissue size limits the number of genes examined. The present study evaluates the Clontech SMART amplification to test the hypothesis that this amplification provides RNA in sufficient quantity and quality to evaluate large numbers of genes in airways < 2 mm diameter obtained by LCM. Commercial reference RNA was amplified 200-fold and the expression levels of 51 genes relative to the unamplified RNA had a correlation coefficient of 0.84. For two pairs of RNA preparations (commercial placenta versus commercial lung; lung sections prepared for LCM from GOLD 0 (at risk for COPD) versus GOLD 2 (moderate disease) patients linear regression of Delta Ct's (delta cycle thresholds) of unamplified versus amplified RNA gave correlation coefficients of R = 0.95. In RNA from microdissected small airways, expression patterns in all GOLD classes of COPD severity were very similar between unamplified and amplified RNA. We conclude that SMART amplification provides cDNA sufficient for studying large numbers of genes even in laser-captured small airways and this cDNA maintains the relative expression found in corresponding unamplified RNAs.
Gosselink
John V
JV
St. 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
Hayashi
Shizu
S
Chau
Edmond
E
Cooper
Joel
J
Elliott
W M
WM
Hogg
James C
JC
D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D018076:DNA, Complementary / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D042282:Microdissection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D029424:Pulmonary Disease, Chronic Obstructive / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D012720:Severity of Illness Index
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D018076:DNA, Complementary; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; C075278:platelet-derived growth factor A; C512478:EGFR protein, human; D066246:ErbB Receptors