The use of gene expression profiling (GEP) in cancer management is rising, as GEP provides insight in molecular and cellular processes through evaluation of thousands of genes at once. GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response and prognosis. However, the reliability of GEP heavily depends on input of RNA at sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from tumor biopsies, which are often small with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue, when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases, to homogenize or to deparaffinize tissues, and the impact of tissue composition on RNA extraction were studied. Additionally, the RNAs compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using RNA and target specific probes of 50 bases that can bind to small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department could be used for RNA extraction. Both resulting RNA end products are compatible to the nanoString nCounter® technology.