Tumor Sample Preparation and DNA/RNA Extraction

Tumor samples from FAIMoS and Edinburgh were microdissected under a stereomicroscope (Olympus SZ61, Tokyo, Japan) using a sterile needle to ensure a tumor cell content >90% as previously described [30 (link)]. DNA extraction was performed by proteinase K digestion and phenol-chloroform precipitation. Total RNA from FAIMoS samples was extracted from un-microdissected sections using the RNeasy kit (Qiagen, Hamburg, Germany) as previously described [31 (link)]. RNA purity and integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA, USA), with samples only being analyzed if RNA integrity scores were >5.0. Sample processing and RNA extraction from samples of the Edinburgh cohort has been previously described [6 (link)]. For cell lines, DNA was extracted using the DNeasy blood and tissue kit (Qiagen) and RNA was extracted using the RNeasy kit (Qiagen).

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López-Knowles E., Wilkerson P.M., Ribas R., Anderson H., Mackay A., Ghazoui Z., Rani A., Osin P., Nerurkar A., Renshaw L., Larionov A., Miller W.R., Dixon J.M., Reis-Filho J.S., Dunbier A.K., Martin L.A, & Dowsett M. (2015). Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer. Breast Cancer Research : BCR, 17(1), 35.

Publication 2015

RNeasy kit Agilent Bioanalyzer DNeasy blood and tissue kit

Blood Cell Cell lines Chloroform Digestion Needle Phenol Proteinase k Sterile Tissue Tumor

Corresponding Organization :

Other organizations : Breast Cancer Now, Institute of Cancer Research, Royal Marsden Hospital, AstraZeneca (United Kingdom), University of Edinburgh, University of Cambridge, Memorial Sloan Kettering Cancer Center, University of Otago

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Variable analysis

independent variables

Microdissection of tumor samples under a stereomicroscope to ensure a tumor cell content >90%

dependent variables

DNA extraction by proteinase K digestion and phenol-chloroform precipitation
RNA extraction from un-microdissected sections using the RNeasy kit
RNA purity and integrity assessment using the Agilent Bioanalyzer

control variables

Use of a sterile needle for microdissection
RNA integrity score threshold of >5.0 for sample analysis
DNA extraction using the DNeasy blood and tissue kit for cell lines
RNA extraction using the RNeasy kit for cell lines

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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