Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection.
Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.
Upson
John J
JJ
Division of Population Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Stoyanova
Radka
R
Cooper
Harry S
HS
Patriotis
Christos
C
Ross
Eric A
EA
Boman
Bruce
B
Clapper
Margie L
ML
Knudson
Alfred G
AG
Bellacosa
Alfonso
A
D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D053685:Laser Therapy; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger