Small rna analysis kit

The small RNA profiles of RNase 1-treated, ANG-treated, and untreated cells were measured with the small RNA gel-electrophoresis chip from Agilent Technologies. Cells were grown to 80%–100% confluency in DMEM containing FBS (10% v/v) and penicillin–streptomycin solution (1% v/v), then counted with a Coulter cell counter and seeded into a flat-bottomed six-well plate at 100,000 cells per well. The cells were incubated for 24 h, after which the medium was replaced with FBS-free DMEM containing RNase 1 or ANG in PBS. Concentrations were chosen to produce ∼75% and ∼50% cell viability, which corresponded to 0.75 and 4 µM treatments of ANG and 1.5 and 18 µM treatments of RNase 1, respectively. After a 48-h incubation, RNA from each well was isolated with the miRCURY Cell and Plant RNA Isolation kit from Exiqon following the manufacturer's instructions. A small aliquot from each sample was then loaded onto a small RNA chip using the Small RNA Analysis kit from Agilent Technologies. Samples were analyzed at the University of Wisconsin Biotechnology Center with a 2100 Bioanalyzer system from Agilent Technologies.

The miRNA quantity was measured using the Small RNA Analysis Kit (Agilent Technologies Ltd) according to manufacturer’s instructions. The chip was run on the Agilent 2100 Bioanalyzer instrument (Agilent Technologies Ltd).

Poly(A)⁺ RNA was isolated using oligo-dT 25-coated polystyrene Dynabeads (Life Technologies) and used for real-time PCR. For total RNA extraction cells were washed with PBS and lysed in Qiazol. The lysate was then loaded into QIAshredder column to break DNA, and small (<200 nucleotides) and long (>200 nucleotides) RNAs were purified using miRNeasy kit (Qiagen) following the manufacturer's instructions. Small RNAs were used for subsequent applications or tRNA purification. RNA was quantified by nanodrop and quality was evaluated by an Agilent Bioanalyzer (Agilent) using the Small RNA Analysis kit.

All the samples were collected within a 1 h after the animals were euthanized, processed immediately, and stored in aliquots at −80 °C until RNA isolation. The total RNA, which included miRNAs, was isolated from each of the individual samples using miRCURY™ RNA Isolation Kits -Biofluids (Qiagen, Germantown, MD). A small aliquot of the sample was frozen separately for quantification and quality control on BioAnalyzer, so the main sample tube was thawed only once during the process of labeling probe for GeneChip microarray hybridization. Total isolated RNA (5–10 ng/μl) from fluid samples was analyzed using the BioAnalyzer using Small RNA Analysis Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer’s protocol.