Rneasy mini protocol

GL261 murine glioma cells (NCI Tumor Repository, Frederick, MD) were grown and passaged in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO₂. GL261 cells were transduced with HIV1-based lentiviral vector plasmid pHRSIN-CSGW-DINotI expressing firefly luciferase (luc) under the control of the spleen focus-forming virus promoter. Lentiviral vectors were generated by transfection of 293 T cells with plasmids encoding the vesicular stomatitis virus G envelope, gag-pol, and luc (generously provided by Dr. Y. Ikeda, Mayo Clinic, Rochester, MN) [13 (link)]. Conditioned medium containing viral vectors were harvested 48 hours post-transfection, filtered (0.45 μm), and frozen until use. GL261 cells were transduced using viral supernatants. Expression of luc was confirmed by measuring luciferase activity (IVIS Lumina imaging station, Caliper Life Sciences), which we have previously demonstrated is highly correlated with cell number [14 (link)]. The transduced cell lines will now be referred to as GL261.luc. RNA was extracted using the RNeasy mini protocol (QIAGEN). RNA concentration was determined by spectrophotometry.

FFPE tumor ribbons from patients with Ewing sarcoma were deparaffinized with Hemo-De, tissue was washed twice in 100% ethanol, and allowed to air dry for 45 minutes. Tissue was digested and RNA was isolated following the Recoverall Total Nucleic Acid Isolation protocol (Ambion Life Technologies). RNA was isolated from cell lines using QIAGEN's RNeasy Mini protocol with QIAshredder (QIAGEN). RNA was treated with DNase using the TURBO DNA-free Kit (Ambion Life Technologies) and quantified using Quant-iT Ribogreen RNA Reagent and Kit (Invitrogen).

Before handling RNA, surfaces and instruments underwent treatment with RNase Zap (Ambion AM9780, Austin, TX, USA). RNA extraction from cultured cells followed the Rneasy Mini protocol (Qiagen 7414, Hilden, Germany). Initially, cells were rinsed with PBS, then lysed with buffer RLT and 10 μL/mL β-mercaptoethanol, utilizing 350 μL per T25 flask or 600 μL per 10 cm plate. Subsequently, the Rneasy Mini protocol, inclusive of on-column incubation with Rnase-free Dnase (Qiagen, Cat: 79254), was adhered to. Post washes, RNA was eluted using 30 μL Rnase-free water, and its concentration was determined employing a Nanodrop spectrophotometer. RNA samples were stored at −80 °C, with each sample being isolated and utilized individually in triplicate. All raw data generated in this study, including FASTQ sequencing data and metadata, have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/ (submitted on 13 May 2024) under accession number GSE267396. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE267396.

RNA was isolated from ∼50 mg of mouse quadriceps tissue in 1 mL of TRIzol (Invitrogen) using a TissueLyser (Qiagen) according to the manufacturer’s instructions. The RNA was resuspended into 50 µL DEPC treated water (Bioline). RNA was purified and DNAseI (Qiagen) treated using RNeasy Mini protocol (Qiagen). Quality and quantity was assessed using a 2100 Bioanalyser (Agilent Technologies) 6000 RNA kit (Agilent Technologies) according to the manufacturer’s instructions. One microgram (1 µg) of RNA was reverse transcribed using Super-Script™ III Reverse Transcriptase (Invitrogen), random pdN6 primers (Roche), 0.1 mM DTT (Invitrogen) and 10 mM dNTP (Invitrogen) in a 20 µL volume for 90 minutes at 50°C on a Veriti 96-well thermocycler (Applied Biosystems). All cDNA was diluted 1∶5 in DEPC treated water (Bioline) prior to RT-qPCR. Except for the Rn18S and Gapdh, the cDNA were diluted to 1∶100 due to the high abundance of these transcripts. Diluted cDNA was aliquoted into working volumes of 10 µl and stored at -80 °C until use. A maximum of three freeze-thaw cycles were performed for each aliquot.

Human tissue samples were obtained from the Cooperative Human Tissue Network, Pediatric Division at Columbus Nationwide Children’s Hospital after institutional review board approval. All specimens were snap-frozen and stored at −80 °C. The tissue was ground using a mortar and pestle in liquid nitrogen. RNA was extracted from tissue samples (25–40 mg) using RNeasy Mini Protocol (Qiagen, Cat # 74106). Typically 1 µg of RNA was used for RT in 20-µl reactions^{28 (link)}. This study received the institutional review board (IRB) approval from the Abigail Wexner Research Institute, Nationwide Children’s Hospital and The Ohio State University.

Total RNAs were extracted from 100 mg liver, kidney and visceral WAT (gonadal) using QIAzol reagent (Qiagen, Hilden, Germany), followed by a purification using RNeasy Mini protocol (Qiagen, Hilden, Germany). Total RNA was treated with DNase I (Macherey-Nagel™, Bottrop, Germany) on mini-columns to eliminate genomic DNA. The RNA quantification was assessed by using the NanoDrop 2000 spectrophotometer (Thermo Fisher, Schwerte, Germany) and RNA quality was determined using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Ratingen, Germany), according to the manufacturer’s instructions.
First-strand cDNA was synthesized from 1 μg of total RNA for each reaction using the SuperScript II Reverse Transcriptase Kit (Thermo Fisher, Schwerte, Germany), according to the manufacturer’s instructions. The cDNA was diluted 1:5 or 1:10 with water, depending on the target gene, and stored at − 20 °C prior to quantitative real-time PCR (qRT-PCR).