Agilent rna 6000 nano kit

Total RNA, including miRNAs, was extracted using a miRNeasy Mini Kit according to the manufacturer’s instructions. RNA concentrations were assessed using the NanoDropTM spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples were immediately frozen at −80 °C until use. The RNA quality was assessed using the Agilent RNA 6000 Nano Kit (5067-1511, Agilent, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies) as per the manufacturer’s instructions. Our samples’ RNA integrity numbers (RIN) ranged from 8.8 to 10, indicating a high degree of RNA integrity. Small RNAs represented 20% of the total RNA, while miRNAs represented 17% of the total small RNAs. We saw no significant changes to these percentages upon treatments with OA and Ex-4.
We used an RNA Broad-Range Assay Kit (Q10211, Invitrogen, Carlsbad, CA, USA) and Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) to measure the RNA concentration and an Agilent RNA 6000 Nano Kit (5067-1511, Agilent, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to assess the RNA quality, as per the manufacturer’s instructions. RNA concentrations ranged from 266.4 to 1680.8 ng/uL. RNA with a 260/230 nm absorbance ratio of >1.8 and 260/280 nm absorbance ratio > 1.8 was used for subsequent experiments on the NanoString nCounter platform.

Prior to miRNA extraction, exosomes were isolated from cell-conditioned media using the miRCURY Exosome Cell/Urine/CSF Kit (Qiagen, Hilden, Germany), and miRNA isolation was performed using the miRCURY RNA Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. To allow for normalization of sample-to-sample variation in the RNA isolation, prior to purification, each sample was spiked with UniSp2, UniSp4, UniSp5, each at a different concentration in 100-fold increments, with UniSp6 and cel-miR-39-3p as positive controls for cDNA synthesis. RNA quality was determined with the Agilent RNA 6000 Nano Kits using 2100 Bioanalyzer (Agilent Technologies, St. Clara, CA, USA). The degradation rate of total RNA was determined using RIN values. Only the samples with RIN > 7 were included in further analysis. Directly after isolation, RNA was subjected to reverse transcription [45 (link)].

RNA was extracted from renal cortex, liver, and epididymal fat pads using an RNeasy Plus Mini Kit (Qiagen). The concentration and purity of RNA samples were determined using a Nanodrop™ 2000 Spectrophotometer (Thermo Fisher Scientific) and RNA sample integrity assessed using Agilent RNA 6000 Nano kits (Agilent Technologies).
For renal cortical RNA sequencing, RNA library preparation was carried out using the TruSeq Stranded Total RNA Library Prep Gold^® kit (Cat. No. 20020598, Illumina). Libraries were sequenced on the Illumina NovaSeq 6000^® platform in a paired-end fashion at a read length of 2x100bp. Sequencing fastq files and raw counts of aligned reads have been deposited in GEO (accession number GSE147706).
For quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses, samples were treated with DNase I and complementary DNA (cDNA) synthesized using SuperScript™ II Reverse Transcriptase Kit (Invitrogen). mRNA expression of Pdk4 in renal cortex and of Acox1, Ehhadh, and Acaa2 in three tissue depots (renal cortex, liver, and epididymal fat) was quantified using beta-actin as the endogenous reference gene (TaqMan^® Gene Expression Assays, Thermo Fisher Scientific and QuantStudio 7 Flex System, Applied Biosystems). Comparative analysis was performed using the ΔΔCt method (41 (link)), with SD animals serving as calibrators.