A molecular test providing clear identification of individuals at highest risk 
for developing metastatic disease from among early stage breast cancer patients 
has proven to be of great benefit in breast cancer treatment planning and 
therapeutic management. Patients with high risk of disease recurrence can also 
get an estimate of the magnitude of benefit to be gained by adding chemotherapy 
to surgery and hormonal therapy. Developing this clinical test was made possible 
by the availability of technologies capable of identifying molecular biomarkers 
from the gene expression profiles of preserved surgical specimens. Molecular 
tests such as the Oncotype DX(®) breast cancer test are proving to be more 
effective tools for individualized patient stratification and treatment planning 
than traditional methods such as patient demographic variables and 
histopathology indicators.Molecular biomarkers must be clinically validated 
before they can be effectively applied toward patient management in clinical 
practice. The most effective and efficient means of clinical validation is to 
use archived surgical specimens annotated with well-characterized clinical 
outcomes. However, carrying out this type of clinical study requires 
optimization of traditional molecular expression profiling techniques to analyze 
RNA from fixed, paraffin-embedded (FPE) tissues. In order to develop our 
clinically validated breast cancer assay, we modified molecular methods for RNA 
extraction, RNA quantitation, reverse transcription, and quantitative PCR to 
work optimally in archived clinical samples. Here, we present an updated 
description of current best practices for isolating both mRNA and microRNA from 
FPE tissues for RT-PCR-based expression profiling.