Reliaprep rna cell miniprep system

RNA extraction was performed on pre-lysed samples stored at −80 °C, using the ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The resulting RNA was measured using a Nanodrop 100 V3.8.1 (ThermoFisher Scientific, Waltham, MA, USA), and then it was stored at −80 °C. RNA was then reverse transcribed using a TaqMan Kit (Applied Biosystems, Waltham, MA, USA) with either 20 µL or 40 µL total reaction volume. The cDNA was then generated using a Verriti 96-well Thermal Cycler (ThermoFisher Scientific) using the following protocol: 10 min at 25 °C, 30 min at 48 °C then 5 min at 95 °C. The cDNA samples were stored at −20 °C, then analyzed by qPCR, with a GoTaq qPCR Master Mix (Promega). Master mix: 10 µL Power Sybr Green MM, 0.75 µL of 5 µM forward primer, 0.75 µL of 5 µM reverse Primer (Sigma, 100 µM stock), and 7.5 µL nuclease free water per well. 18 µL of the master mix was combined with 2 µL of cDNA, and qPCR was performed in a 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA), then it was analyzed using Applied Biosystems 7500 System SDS Software v2.0.5. A list of primers is provided in Table 2.

Transfected HEK293T cells were incubated in SF-DMEM (3 h) before treatment without or with OSM (10 ng/ml, 45 min for socs3, 60 min for c-fos) and RNA extraction (ReliaPrep RNA Cell Miniprep System, Promega). cDNA was generated using oligo(dT)₂₀ primer (GoScript Reverse Transcription System, Promega), before qPCR using primers for socs3, c-fos and gapdh, and iTaq Universal SYBR Green Supermix (Bio-Rad). Standard curves were generated for each primer pair using serial dilutions of the reference cDNA (samples from GFP-N-protein-expressing cells treated with OSM). Values were normalised to gapdh [6 (link)] and then calculated relative to that determined for untreated GFP-N-protein-expressing cells. Data from 3 independent assays were combined, where each assay result is the mean of replicate samples. Primers sequences were: 5’-GGTGCATTACAGAGAGGAGAAA-3’ and 5’ GTGTGTTTCACGCACAGATAAG-3’ for c-fos; 5’-GGAGTTCCTGGACCAGTACG-3’ and 5’-TTCTTGTGCTTGTGCCATGT-3’ for socs3; 5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GGTCATGAGTCCTTCCACGAT-3’ for gapdh.

Changes in relative gene expression were analysed by RT-PCR. EA.hy926 cells were cultured in 6-well plates (Corning TM, Corning, NY) (cell density of 6 x 10⁵ cells/mL) with FAs for 48 h followed by incubation with TNF-α (1 ng/mL) for 6 h. Taqman Gene Expression Primers (ThermoFisher Scientific, Waltham, MA, USA) were used to determine the expression of NFκB1 (Hs00765730_m1), NFκBIA (Hs00355671_g1), IκBKB (Hs00233287_m1), PTGS2 (Hs00153133_m1), and IL-6 (HS00985639_m1). Total RNA was extracted from the cells using the ReliaPrep RNA cell Miniprep System (Promega, Southampton, UK). RNA quantity and quality were analysed by NanoDrop. Analysis of RNA using an Agilent Bioanalyzer (RNA Total Eukaryote 2100 Nano) was performed to determine RNA integrity through RIN scores. cDNA was synthesised from total RNA using GoScript Reverse Transcriptase (Promega). Housekeeping reference genes were determined using a geNorm Kit (Primerdesign, Camberley, UK). Quantification of relative gene expression was analysed using YWHAZ, (Hs01122445_g1), CYC1 (Hs00357717_m1), and RPL13A (Hs04194366_g1) as housekeeping genes.

Total RNA was extracted from freshly frozen tumor samples using the ReliaPrep RNA Cell Miniprep System (Promega, USA) and reverse-transcribed using random hexamers and the Transcriptor High Fidelity cDNA synthesis kit (Roche, Germany) as per the manufacturer’s instructions. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (Roche, USA) with previously-published primer sets^{32 (link)} on a ViiA^TM 7 Real-Time PCR System (Applied Biosystems, USA).

Total cellular RNA was extracted using the ReliaPrep RNA Cell Miniprep System (Promega) and treated with DNase I according to the manufacturer’s protocol. Quantity of the purified RNA was measured spectrophotometrically (A260/A280) using NanoPhotometer (Implen, Germany). Quality of isolated RNA was assessed by capillary electrophoretic measurement of RNA integrity number (RIN) using Bioanalyzer 2100 (Agilent, Palo Alto, CA). Samples with RIN values exceeding 9 were taken for further analyses.

RGs were dissected from wandering third instar larvae for two wild-type strains: the reference genome strain y¹; cn¹ bw¹ sp¹ (Cel) and Armenia¹⁴ (A14) (Perry et al. 2012 ) (all fly stocks listed in Supplemental Material, Table S1). Dissections were performed in 100% PBS in batches of 10–40 at a time, then pooled into three biological replicates for both Cel and A14; ∼80 RGs were pooled to provide the ∼1 μg of RNA required for sequencing. Total RNA was extracted using the Reliaprep RNA Cell Miniprep System (Promega), then stored at −80°. Total RNA was quality assessed using the 2100 Bioanalyzer (Agilent Technologies), polyA enriched, cDNA libraries prepared, and 100 bp paired-end RNA-seq performed on the Illumina HiSeq2000 system (Australian Genome Research Facility, AGRF). In addition to the reads obtained from the six RG samples, duplicate RNA-seq reads for the Oregon-R wandering third instar CNS were downloaded from the modMINE database (accession: SRX029398) (Contrino et al. 2012 (link)). These reads were downloaded in SRA (short read archive) format, and converted to paired end fastq format using the fastq-dump utility included in the NCBI SRA toolkit.