Rt2 pcr array first strand kit

MCF-7 or LCC9 cells were serum-starved, as above, for 48 h and then treated with DMSO (vehicle control), 10 or 50 μg/ml β-D-glucan, 10 nM E₂, or 100 nM 4-OHT. Total RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA quality was examined by NanoDrop Spectroscopy and cDNA synthesis was performed using the RT2 PCR Array First Strand kit (SABiosciences, Qiagen). RT2 Profiler PCR Array Breast Cancer SABiosciences cat no. PAHS-131ZA-12 contains 84 genes commonly involved in the dysregulation of signal transduction and other biological processes during breast carcinogenesis and in breast cancer cell lines plus 5 housekeeping genes http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-131Z.html. Breast cancer PCR arrays were performed according to the manufacturer’s instructions. Data analysis was performed using the web-based analysis tool (www.sabiosciences.com/pcrarraydataanalysis.php), including fold change and cluster analyses.

QH (Barrett’s) and GO (dysplasia) cell lines, representing the Barrett’s and HGD stages of the Barrett’s disease sequence, were grown to 70% confluency in BEBM medium (2 mM glutamine, 10% FBS, 1% penicillin-streptomycin l-glutamine) supplemented with BEBM SingleQuots (2 mL BPE, 0.5 mL insulin, 0.5 mL HC, 0.5 mL GA-1000, 0.5 mL retinoic acid, 0.5 mL transferring, 0.5 mL triiodothyronine, 0.5 mL adrenaline and 0.5 mL hEGF per 500 mL media). OE33 cells, representing OAC, were grown to 70% confluency in RPMI medium (2 mM glutamine, 10% FBS, 1% penicillin-streptomycin l-glutamine). QH (or CP-A) and GO (or CP-B) cell lines were obtained from American Type Culture Collection (ATCC) (LGC Standards, Middlesex, UK). The OE33 cell line was sourced from the European Collection of Cell Cultures (Sailsbury, UK). Cell lines were authenticated and characterized by the suppliers and the suppliers use morphology, karyotyping and PCR-based approaches to confirm the identity of cell lines. Cell RNA extractions were subsequently performed using RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. RNA content and quality was quantified and assayed respectively and RNA reverse transcribed using the RT² PCR array first strand kit (SABiosciences, Frederick, MD, USA).

Total RNA (1.0 μg) was extracted with TRIzol and quantified using NanoDrop Spectrophotometer (Thermo Scientific NanoDrop Technologies, Wilmington, DE, USA). Reverse transcription reaction was prepared with the RT² PCR array first strand kit from SABiosciences (Qiagen Company). Real-time PCR was conducted to evaluate 84 genes for chemokines and receptors RT² Profiler PCR array of rat (PARN-022Z, Qiagen).
The amplification assays were made using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) (http://www.sabiosciences.com/PCRArrayPlate.php).