Quantifluor rna system kit

To extract RNA from isolated EVs, QIAzol lysis reagent (QIAGEN AG, Hombrechtikon, Switzerland) followed by miRNeasy micro kit (QIAGEN) was used according to the manufacturer´s instructions. RNA concentration was measured by different RNA quantification methods: Agilent RNA 6000 Pico assay (Agilent 2100 Bioanalyzer, Agilent Technologies Schweiz AG, Basel, Switzerland) for RNA quantity and quality profiles of EVs samples; and (2) Quantus™ Fluorometer (Promega AG, Dübendorf, Switzerland) together with QuantiFluor RNA System kit (Promega).

Extraction and isolation of microRNA were performed using the Maxwell^® RSC miRNA Plasma and Serum Kit (Promega) on the Maxwell^® RSC Instrument (Promega), following the manufacturer's instructions, using 300 μL of plasma in each extraction. The sample quantification was estimated using the Quantus™ Fluorometer, pre-programmed with RNA quantification protocols using the QuantiFluor^® RNA System kit (Promega Corporation, USA).
To convert the extracted RNA into cDNA, the TaqMan^® Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific) was used, following the manufacturer's instructions. In addition, ddPCR reaction was performed for miR-21 evaluation, where a CNV reaction was performed using pre-designed master mix and probes (Bio-Rad, USA) on the QX200 Droplet Digital PCR System.

Total RNA was extracted from Trizol-stabilized cell pellets using NucleoSpin 96 miRNA kit (Macherey-Nagel). RNA concentrations were measured using Quantifluor RNA system kit (Promega) and RNA integrity numbers were determined using the Agilent RNA 6000 Nano kit (Agilent Technologies). Total RNA samples were analyzed using the Human Host Response panel profiling 800 immunology and host response-related human genes (Nanostring). Gene expression data were normalized as previously described using nCounter software (Nanostring)^{39 (link)}.

Total RNA was extracted from Tregs derived from the BM of naive and CML-bearing Foxp3^DTR/GFP mice (n = 3/group) using the RNeasy Micro Kit (catalog 74004, Qiagen). Total RNA quality was determined by a Bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies) and quantified by fluorometry using the Quantifluor RNA System Kit (catalog E3310, Promega) on a Quantus Fluorometer Instrument (Promega).
Library preparation was performed from total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). Libraries were quality checked on the Fragment Analyzer using the High Sensitivity NGS Fragment Analysis Kit (Agilent). Samples were pooled to equal molarity, and the pool was quantified by fluorometry, in order to be loaded at a final concentration of 2 pM on the NextSeq 500 instrument (Illumina). Samples were sequenced SR76 using the NextSeq 500 High Output Kit 75-cycles (Illumina), and primary data analysis was performed using the Illumina RTA version 2.4.11 and bcl2fastq v2.20.0.422.

RNA extraction was performed using a miRNeasy Serum/Plasma Kit (Qiagen, Germany) in line with the manufacturer’s instructions. RNA concentrations and purity were calculated using Quantifluor Handheld Fluorometers E6090 (Promega, USA) with a Quantifluor RNA System Kit as specified by the manufacturer.

25 to 30 mg of liver tissue from all groups were processed with the Aurum Total RNA kit (Biorad No. 732-6820). First, the tissue was homogenized with 700 µL of lysis buffer (guanidinium thiocyanate and β-mercaptoethanol) and transferred to a 1.5 mL solvent-resistant conical microtube. The sample was centrifuged at 13,000 rpm for 5 min at room temperature. Subsequently, the supernatant was transferred to a new tube, and 700 µL of 70% ethanol was added and homogenized. Next, 700 µL of the mixture was transferred to an RNA affinity column and centrifuged at 13,000 rpm. After centrifugation, 700 µL of low stringency buffer was added to the column and then centrifuged at 13,000 rpm. Then, 80 µL of DNase was added to the column, and the sample was incubated for 15 min at room temperature. After incubation, a second wash was performed with 650 µL of high stringency buffer, centrifuged at 13,000 rpm. Finally, a last wash was performed with 700 µL of low stringency buffer and centrifuged at 13,000 rpm, followed by dry centrifugation at 13,000 rpm. To perform the elution, 20 µL of water free of RNAsas was used. Subsequently, the RNA concentration was quantified using the Quanti Fluor RNA System kit in a Quantus fluorometer (Promega). Finally, the RNA quality was analyzed in a QIAxpert high-speed UV/VIS microfluidic spectrophotometer (Qiagen).