Rna nano chip kit

Total RNA was isolated using the Illustra RNAspin Mini kit (GE Healthcare Life Sciences, Buckinghamshire, UK). Approximately 5 × 10⁶ cultured cells were processed following the manufacturer's instructions. Samples were eluted in Ultrapure DNase/RNase-free distilled water, which was provided in the kit. RNA samples were quantified using ultraviolet spectroscopy (NanoDrop, Wilmington, DE) and were further assessed for RNA integrity (RIN) on the Aglient 2100 Bioanalyzer (Santa Clara, CA) using the RNA Nano-chip Kit. RNA samples with RIN values of seven or better were used for further analysis.

Total RNA was extracted from rib cartilage for microarray analysis. Thoracic cage of P4 mice was dissected, digested with 2 mg/ml collagenase type II in DMEM at 37 °C for 90 min and costal cartilages were harvested from each rib under the dissection microscope. The RNA then was purified from costal cartilage using RNeasy Plus Mini kit (QIAGEN) according to manufacturer's protocol. RNA quality was analysed by Agilent 2100 Bioanalyzer (Agilent Technologies) using the RNA Nano chip kit (Agilent Technologies), while RNA concentration was determined by the NanoDrop ND-1000 spectrophotometer.
The GeneChip® Mouse Genome 430A 2.0 microarray (Affymetrix) was used and processed with the GeneChip® 3’ IVT PLUS Reagent Kit and the GeneChip® Hybridisation Wash and Stain Kit. The microarray analysis was performed by the Center for Genome Research of the University of Modena and Reggio Emilia, Italy. The fold change threshold between Cant1^−/− and Cant1^+/+ mice was set ±2.

RNA of treated and control HROG63 cells (5 × 10⁵ cells/treatment) were extracted employing RNeasy Plus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The total RNA was quantified on a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific) and integrity confirmed using the Agilent Bioanalyzer 2100 with an RNA Nano chip kit (both from Agilent Technologies, Waldbronn, Germany). Expression profiling was performed by taking advantage of the Affymetrix Human Clariom S Array (Affymetrix/Thermo Fisher Scientific, Santa Clara, USA), which interrogates over 20,000 well-annotated genes. Therefore, the so-called Whole Transcriptome protocol was employed described in ref. ^{58 (link)}. Primary data analysis was performed with the Affymetrix Transcriptome Analysis Console (TAC) software, including the SST-RMA for normalization. Gene expression data were log-transformed. Limma was used here to calculate the P value. A change was considered significant when the Limma eBayes P value met the criterion P < 0.05 at fold changes >|2|, i.e., expression increments or declines larger than two.

Tumors from patients with ovarian cancer (serous and endometrioid adenocarcinoma) were collected and stored at –80°C for later use. Tumors weighing 30–40 mg were placed in 750 µL Invitrogen TRIzol (Thermo Fisher Scientific) and cut into smaller pieces with scissors. To each sample 150 µL of chloroform was added, and samples were mixed for 30 seconds and centrifuged at 20,854 ×  g for 15 minutes at 4°C to separate RNA into the aqueous phase. The aqueous phase was mixed with approximately 0.53 × volume of 100% ethanol and transferred to an RNeasy spin column (Qiagen). Total RNA was purified with RNeasy Mini kit (Qiagen) as per manufacturer's protocol with RNAse free DNase (Qiagen) on-column treatment. RNA concentration and integrity were measured on the 2100 Bioanalyzer (Agilent Technologies) using the RNA NanoChip Kit (Agilent).

Following assessment of the quality of total RNA using Agilent 2100 bioanalyzer and RNA Nano Chip kit (Agilent Technologies, CA), rRNA was removed from 2.5 µg of RNA with Ribo-Zero rRNA removal kit for Gram-negative bacteria (Epicentre Biotechnologies, WI). To generate directional signal in RNA seq data, libraries were constructed from first strand cDNA using ScriptSeq v2 RNA-Seq library preparation kit (Epicentre Biotechnologies, WI). Briefly, 50 ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. Following purification by a magnetic-bead based approach, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences and add adaptor sequences required for sequencing cluster generation. Amplified RNA-seq libraries were purified using AMPure XP System (Beckman Coulter). Quality of libraries were determined via Agilent 2100 bioanalyzer using DNA High Sensitivity Chip kit, and quantified using Kappa SYBRFast qPCR kit (KAPA Biosystems, Inc, MA). 50 bp sequence reads were generated using the Illumina HiSeq 2000 platform.

Total RNA was isolated from frozen samples using miRCURY RNA (Exiqon). RNA integrity number (RIN) was verified for each sample using a RNA Nano Chip Kit (Agilent Technologies, Germany) in Agilent Bioanalyzer 2100.