This document discusses using xMD technology to analyze microRNA in triple negative breast cancer samples. The goal is to identify new biomarkers for triple negative breast cancer. The general procedure involves immunohistochemistry on tumor sections, expression microdissection to isolate tumor cells, miRNA isolation, reverse transcription of miRNA to cDNA, and real-time PCR analysis. xMD specifically isolates tumor cells, providing a more accurate analysis than traditional macrodissection. Identifying abnormalities in tumor cells' microRNA could help prescribe targeted medications to treat triple negative breast cancer.

1. MicroRNA Analysis of Triple
Negative Breast Cancer Using xMD
Technology
Molly Hulseman

2. Goal Of Research
• What we know: microRNA (miRNA)
dysregulation has shown strong correlation
with metastatic cancers
• To identify potential new biomarkers of Triple
Negative Breast Cancer (TNBC) that could
ultimately improve molecular diagnostics and
patient care

3. General Procedure
• 1. Immunohistochemistry on patient tumor
sections
• 2. Expression Microdissection (xMD)
• 3. miRNA isolation
• 4. Reverse transcriptase of miRNA to cDNA
• 5. Real-Time PCR analysis

4. Immunohistochemistry (IHC)
• Process of detecting antigens on the cell
surface of specific cells.
∧ ∧∧ ıı ı∧ ∧∧ ıı ı
CYTOPLASM
(Tumor epithelial cell)
AGAGAG
AG- Antigen
Primary Ab
Secondary Ab
HRP - stain
HRP
HRP HRP

5. xMD
• Today, manual
macrodissection is used to
enrich for tumor cells
• Picks up extra,
noncancerous cells
• May distort results of
molecular analysis
• xMD specifically isolates
the cells of interest
• More accurate assay
readings

6. xMD
• Using a broad spectrum flash lamp and EVA films
• The “tagged” cells adhere to the EVA film and are pulled off
the tissue

7. xMD Films: Isolated Cells
Breast Cancer – Primary Tumor Breast Cancer –Metastatic Tumor

8. miRNA
• Present in the cytoplasm of the cell
• Degrades mRNA, disrupting cells messages

9. Reverse Transcriptase
• Converts miRNA to cDNA in order to go
through PCR amplification.

10. Real-Time PCR
• miRNAs in question : miRNA 181a, 221, 222, 708, and 454

11. xMD can be used to improve
molecular diagnostics
• By microdissecting specifically tumor cells,
pathologists can better identify abnormalities
• Further, can prescribe the proper medication
to treat the specific abnormalities in those
cancer cells
• Proteins expressed by cancer cells can be
targeted by specific medications