microRNA and protein expression in breast cancer formalin-fixed paraffin-embedded samples

Introduction

microRNAs (miRNAs) constitute a new class of small noncoding RNAs that control post-transcriptionally the expression of gene products, either modulating directly protein translation or regulating the stability of messenger RNA. There is increasing evidence of the role that miRNAs play in regulating breast cancer gene expression. The main objective of this study is to evaluate the diagnostic utility of the expression of a panel of miRNAs in breast cancer and compare their expression with the expression of the proteins they regulate.

Methods

miRNA expression was analyzed by RT-qPCR using TaqMan Arrays (Applied Biosystems). We compared the expression of 667 miRNAs on 19 fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) matched breast cancer samples. Regarding protein expression, we have developed and evaluated different protocols for protein extraction from FFPE samples. Next, we studied the applicability of these protein extracts to classical and new high-performance proteomics techniques.

Results

After proper normalization, 123 out of 671 miRNAs showed a good correlation of their expression data between FFPE and FF tissue, and sufficient analytical robustness (they are expressed in at least one-third of FFPE samples). In addition, we analyzed the expression of various markers with diagnostic value in breast cancer. As regards high-performance proteomics, the protocols developed generated over 6,000 MS/MS spectra, enabling the identification of hundreds of proteins in each sample.

Conclusion

We have selected the most appropriate assays to study miRNA expression in breast cancer FFPE archived samples. The protocols developed allow proteome analysis of FFPE samples using the latest mass spectrometry equipment. The technologies implemented during the development of this project allow one to compare the expression data at both miRNA and protein levels to study breast cancer from an authentic system-biology perspective.