Agilent rna 6000 nano chips kit

RNA was collected and extracted using a miRNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) from PBMCs. RNA integrity and quality were controlled with Agilent RNA 6000 Nano Chips kit^® (5067-1511, Agilent, Santa Clara, CA, USA). Reverse transcription was performed with SYBR^® Premix Ex Taq^TM (Takara, Kusatsu, Japan), and cDNA was subjected to quantitative real-time PCR using primers for Htr7 (#QT00012481, Qiagen) and ONEGreen^® Fast qPCR Premix (Ozyme, Saint Cyr l’Ecole, France). Relative RNA expression was normalized to Hprt1 and Gapdh expressions (Qiagen), and raw data were analyzed by the 2^∆∆Ct method [14 (link)].

RNA extractions from the LCLs pellets were performed with the Nucleospin^® RNA kit (Macherey-Nagel, Hoerdt, France) according to the supplier’s protocol. The DNased RNAs obtained were assayed with Nanodrop and the quality was checked using with the Agilent RNA 6000 Nano Chips kit (Agilent Technologies, Santa Clara, CA, USA) on Bioanalyzer 2100. cDNAs were synthesized using the AffinityScript cDNA Synthesis Kit (Agilent Technologies) according to the manufacturer’s recommendations, and regions of interest of cDNAs were amplified using the Taq DNA Polymerase Invitrogen kit (Invitrogen^TM, Waltham, MA, USA). The PCR products were separated on a 1.5% agarose gel at 120V for 1 h. Quantification of bands on agarose gel were carried out with the Image Lab 6.1 software (Bio-rad). PCR products were purified and sequenced by the Sanger method with the 3730XL DNA Analyzer (Applied Biosystems^TM, Waltham, MA, USA). The sequences were read using the SeqScape™ software (Applied Biosystems™).