RNA extraction Total RNA including miR from the tissue samples and cultured cells was extracted using a commercial kit (mirVana RNA? Isolation kit, Applied Biosystems) according to the supplier’s selleck chemical Nutlin-3a instructions. Quality of total RNA was determined on a Bioanalyzer (Bioanalyzer RNA Nano kit, Agilent, Santa Clara, CA), and the RNA was quantified using a Nanodrop-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Extracted RNA samples were stored at -80��C until used. MiR array hybridization and analysis To find specific miR(s) for ESCC cells, total RNA was extracted from OE21 and TE10 cells, representative well and moderately differentiated human ESCC cell lines, respectively, and the non-malignant human esophageal squamous cell line, Het-1A.
The isolated RNA samples were subjected to comprehensive analysis of miRNA expression patterns with the microarray-based technology, an Agilent Human miRNA array chip version 1 (Agilent), containing 15,000 probes corresponding to 470 unique human miRs and 64 human viral miRs cataloged in the Sanger database version 9.1. One hundred ng of each total RNA aliquot were treated with calf intestine phosphatase (GE Healthcare, Chalfont St Giles, UK), denatured using DMSO (Sigma, St Louis, MO), and directly labeled with Cy3 using T4 RNA ligase (GE Healthcare). Labeled samples were hybridized to the miR array 8 �� 15 k (G4470A) platforms in SureHyb chambers (Agilent), washed with the buffer supplied (Agilent), according to the manufacturer’s instructions, and scanned using an Agilent Scanner (G2505B).
Data were extracted using Feature Extraction Software 9.3 and GeneSpring software (Agilent). To identify miRs that were differentially expressed between the ESCC cell lines and Het1A cells, supervised analysis was performed using significance analysis of microarrays (SAM, Stanford University, Stanford, CA). The differences in miR expressions were considered significant if the fold change of expression values was >2.0 and the p value was < 0.05 using the t-test. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for miRs Expression levels of miRs that showed significant differences based on the microarray results were analyzed by quantitative RT-PCR using various human malignant cell lines including ESCC and non-malignant Het-1A.
cDNA was prepared from total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Predesigned TaqMan MicroRNA Assays including the primer set and TaqMan probe were purchased from Applied Biosystems. The reverse Drug_discovery transcription reactions were performed in aliquots containing 50 ng total RNA,1.5 ��l 1 �� RT Primer, 1 ��l 10 �� RT Buffer, 0.15 ��l 100 mM dNTP,1 ��l reverse transcriptase, and nuclease-free water added up to 15 ��l at 16��C for 30 min, followed by 42��C for 30 min and 85��C for 5 min. All PCR reactions were performed in 20-��l aliquots containing 1.33 ��l miR RT products with 18.
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