University of Louisville
Laser Capture Microdissection of Normal & Neoplastic Cells for Gene and Protein Expression
Institution
University of Louisville
Faculty Advisor/ Mentor
James L. Wittliff
Abstract
Molecular basis of clinical behavior and therapeutic response of human carcinoma cells is poorly understood. Cellular heterogeneity and improper tissue handling of clinical specimens has been a complicating factor for assessing analyte/biomarker levels in specific cell types. Our goal is to determine the relationship of gene and protein expression profiles to patient-associated characteristics, tumor pathology & biomarker status and clinical course in human carcinomas to arrive at a new classification, to assess patient prognosis and to improve therapy selection while monitoring therapy response. Laser Capture Microdissection (LCM) allows procurement of pure populations of various cell types. In preliminary LCM experiments, H & E stained tissue sections from ovarian and uterine biopsies were used to collect 3000-5000 cells. Total RNA was extracted, purified, and the mRNA amplified to compare quantities in whole tissue sections compared to that of LCM procured cells using established protocols. Comparison was also made between samples incubated with and without a nucleic acid carrier (polyinosinic acid). Universal reference RNA served as a control. RNA yield from whole uterine sections (5-6 µm) ranged from 30-290 ng, while LCM procured cells gave 18-79 ng. LCM procured cells of ovarian tissue sections yielded 9-86 ng. Using HSP70 as a model protein, Western Blot analyses required ~10,000 LCM pulses (30 µm spot setting at 75 mW) of endometrial and ovarian tissue sections for protein detection. Preliminary studies suggest the LCM approach allows generation of cell-specific gene and protein expression results for correlation with patient characteristics and cancer clinical behavior.
Laser Capture Microdissection of Normal & Neoplastic Cells for Gene and Protein Expression
Molecular basis of clinical behavior and therapeutic response of human carcinoma cells is poorly understood. Cellular heterogeneity and improper tissue handling of clinical specimens has been a complicating factor for assessing analyte/biomarker levels in specific cell types. Our goal is to determine the relationship of gene and protein expression profiles to patient-associated characteristics, tumor pathology & biomarker status and clinical course in human carcinomas to arrive at a new classification, to assess patient prognosis and to improve therapy selection while monitoring therapy response. Laser Capture Microdissection (LCM) allows procurement of pure populations of various cell types. In preliminary LCM experiments, H & E stained tissue sections from ovarian and uterine biopsies were used to collect 3000-5000 cells. Total RNA was extracted, purified, and the mRNA amplified to compare quantities in whole tissue sections compared to that of LCM procured cells using established protocols. Comparison was also made between samples incubated with and without a nucleic acid carrier (polyinosinic acid). Universal reference RNA served as a control. RNA yield from whole uterine sections (5-6 µm) ranged from 30-290 ng, while LCM procured cells gave 18-79 ng. LCM procured cells of ovarian tissue sections yielded 9-86 ng. Using HSP70 as a model protein, Western Blot analyses required ~10,000 LCM pulses (30 µm spot setting at 75 mW) of endometrial and ovarian tissue sections for protein detection. Preliminary studies suggest the LCM approach allows generation of cell-specific gene and protein expression results for correlation with patient characteristics and cancer clinical behavior.