![]() ![]() The arrays were scanned using Agilent’s Microarray Scanner (Model #G2565A). Arrays were washed first at room temperature with Agilent’s Gene Expression Wash Buffer #1 and then at elevated temperature (~31º C) using Gene Expression Wash Buffer #2. Samples were loaded on to 4x44K custom Human Ion Channel SpliceArrays (ExonHit Therapeutics, Gaithersburg, MD) and hybridized for 17 h at 65º C using Agilent Sure-Hyb chambers. The C圓-labeled cDNA (5 μg) was incubated at 98✬ for 2 min in the presence of 10X blocking agent (Agilent Technologies, Wilmington, DE) and mixed with 2X HI-RPM Hybridization Buffer (Agilent). The samples were then denatured at 70✬ for 10 min and purified by Microcon YM-30 Filter device. The 3'-end non-bias proportional RNA amplification was carried out using the TransPlex™ Whole Transcriptome Amplification kit (Rubicon Genomics, Ann Arbor, MI).Įach sample was incubated in the dark at 37✬ for 1 h with 120 U Terminal Deoxynucleotidyl Transferase Recombinant Enzyme and Terminal Transferase Buffer (Promega, Madison, WI), and 2.4 nmol Cy-3 dUTP (Amersham Biosciences, Little Chalfont Buckinghamshire, England). ![]() ![]() Cells were laser-capture microdissected with the Arcturus PixCell II system and collected.Ĭellular RNA was extracted using the PicoPure RNA isolation kit (Arcturus). Ventricular myocytes were identified in serial 7-8-µm thick cryostat sections using the RNA-preserving quick (10 s) staining by Eosin Y (Sigma-Aldrich, St. Samples were washed in 4☌ saline, immersed in Tissue-Tek OCT compound (EMS, Hatfield, Pennsylvania), flash frozen in liquid nitrogen and stored at -80˚C until use. LLeft ventricle tissue samples were obtained from material resected from the explanted heart of an ischemic cardiomyopathy patient undergoing cardiac transplantation according to routine clinical protocols. Tissue status: freshly resected tissue sample GEO help: Mouse over screen elements for information. ![]()
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