Breast tumor samples (Table 1) were obtained from patients undergoing surgery after informed written consent (Apollo Hospital; Chennai, India). The excised tumor specimen were immediately preserved in RNAlater (Sigma-Aldrich, MO, USA) and stored at 4 ��C until shipment. All tumor samples utilized in this study were invasive ductal carcinoma. The receptor status of the tumors was determined http://www.selleckchem.com/products/baricitinib-ly3009104.html at the hospital using immunohistochemistry (Supplementary Table 1). Thirty one tumor samples [15 ER�� (+) and 16 ER�� (?)], for which sufficient total RNA could be obtained (~20 ��g total RNA), were used in the microarray analysis and additional 45 tumor samples [31 ER�� (+) and 14 ER�� (?)] were utilized in RT-qPCR studies. Table 1. Clinicopathological characteristics of breast cancer patients.

RNA isolation and reverse transcription The procedure followed was as described by others.19 Briefly, 30 to 50 mg tissues were homogenized in 1 ml of TRIzol (Invitrogen Corporation, CA, USA). The samples were spun at 13,000 rpm for 15 mins at 4 ��C. Subsequently, the upper aqueous layer was collected and purified using RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of RNA samples was determined using NanoDrop ND-1000 spectrophotometer (NanoDrop products, DE, USA). RNA quality was determined using RNA 6000 Nano Lab-on-a-Chip kit (Agilent Technologies, Santa Clara, CA) on the Bioanalyzer 2100 (Agilent Technologies). RNA samples were reverse-transcribed in a total volume of 20 ��L using 200 units of reverse transcriptase, 50 pmol of random hexamer, and 10 mM of deoxynucleotide triphosphates (Invitrogen).

Gene expression profiling Microarray based gene expression data was generated by using 35 K human oligo chip based on 70-mer oligonucleotides (Operon, AL, USA). Fifteen ��g of each tumor RNA and human universal reference RNA (Stratagene, CA, USA) was labeled with Cy5 and Cy3 (GE Healthcare, UK), respectively, according to the protocol established by Pronto Indirect Labeling kit (Promega Corporation, WI, USA). The labeled cDNA concentration and Cy5, Cy3 incorporation were assessed with NanoDrop spectrophotometer. The spotted aminosilane slides were pre-hybridized and post-hybridized according to Universal Micro-array Hybridization kit (Corning Incorporated, NY, USA). Hybridization was run for 6 hrs at 42 ��C, 6 hrs at 35 ��C, and 6 hrs at 30 ��C.

Hybridized slides were scanned using the GeneTAC GT UC scanner (Genomic Solutions Inc., USA). GeneTAC Integrator software was used to analyze the image followed by global normalization. The microarray data were evaluated Drug_discovery using GeneSpring Software (Agilent Technologies, Santa Clara, California). RT-qPCR Template cDNA were synthesized from total RNA isolated from tumor samples. All the PCR reactions were performed using the QuantiFast SYBR Green PCR Kit (Qiagen GmbH) as described by others.