Gene transcript assay by real-time RT-PCR in epithelial breast cancer cells selected by laser microdissection.
The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERalpha, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor homogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.
Becette
V
V
Department de Pathologie, INSERM E0017, Centre René Huguenin, Saint-Cloud, France. v.becette@stcloud-huguenin.org
Vignaud
S
S
Régnier
C
C
Labroquére
M
M
Fourme
E
E
Menet
E
E
Bièche
I
I
Spyratos
F
F
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008875:Middle Aged; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis; D011980:Receptors, Progesterone / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D014408:Biomarkers, Tumor; D017931:DNA Primers; D047628:Estrogen Receptor alpha; D012313:RNA; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011980:Receptors, Progesterone