Qubit rna iq assay kit

All work surfaces and laboratory equipment used for RNA extraction were treated with RNaseZapTM RNase Decontamination Solution (Invitrogen, Waltham, MA, USA). Cell growth media was removed from each well of 6-well plates, and cells were washed with ice-cold PBS and immediately lysed using 1 mL of QIAzol^® Lysis Reagent (Qiagen, Hilden, Germany). Total RNA extracting and genome DNA elimination were performed with the RNeasy^® Plus Universal Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The quantity and quality of the extracted RNA were evaluated using a NanoDropTM 2000 Spectrophotometer (ThermoScientific, Waltham, MA, USA) by measuring the light absorbance at 280 nm, 260 nm, and 230 nm and calculating the 260/280 and 260/230 ratios. RNA integrity was determined by measuring the RNA Integrity and Quality (RIQ) Index using Qubit RNA IQ Assay Kit (Invitrogen, USA) and the Qubit 4 Fluorometer (Invitrogen, USA). The extracted RNA was stored at −80 °C.

Analysis of the expression of tendon-, cartilage- and bone-related genes was performed via RT-qPCR. Given the potentially inhibitory effects of carrageenan (CR) in the reverse-transcription reaction [50 (link)], +MMC groups were not included in the analysis. At the end of all experimental time points, culture media were removed and total RNA was isolated with PureZOL™ RNA isolation reagent. Total RNA concentration and purity were estimated using a SmartSpec™ Plus spectrophotometer (Bio-Rad Laboratories, USA). RNA integrity was assessed using Qubit™ RNA IQ assay kit with a Qubit™ 4 fluorometer (Invitrogen, USA). RNA was reverse-transcribed using iScript™ gDNA Clear cDNA Synthesis Kit. Expression of the tendon-related genes COL1A1, COL3A1, SCX, TNMD and MKX, as well as the chondrogenic and osteogenic genes SOX9 and RUNX2, respectively, was evaluated by real-time PCR using the SsoAdvanced™ Universal SYBR® Green Supermix in a Chromo4™ System connected to a DNA Engine® thermal cycler (Bio-Rad Laboratories, USA). Manufacturer's instructions were followed in every case. Gene expression values were calculated relative to the expression levels of each corresponding gene in hTCs at day 4 by means of the ΔCt method. Primer sequences and characteristics are detailed in Supplementary Table S2.

Before RNA extraction, 1 mL of sorbitol wash buffer (100 mM Tris-HCl pH 8.0 (Sigma-Aldrich), 0.35 M sorbitol (Sigma-Aldrich), 5 mM EDTA pH 8.0 (Sigma-Aldrich), 1% (w/v) polyvinylpyrrolidone (M_W: 40,000, Sigma-Aldrich), and 1% (v/v) 2-mercaptoethanol (Sigma-Aldrich) added just before extraction) was added to 50 mg of macerated plant material, mixed using a vortex, and centrifuged at 5000× g for five minutes at room temperature. The supernatants were discarded and the total RNA was extracted using a Quick-RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) following the supplier’s instructions. The final concentration of RNA of each sample was measured using a spectrophotometer (NanoDrop One, Thermo Scientific, Waltham, MA, USA) and its purity was confirmed using the A260/A280 and A260/A230 ratios. The RNA integrity was further validated using the Qubit™ RNA IQ Assay Kit (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was generated using the NZY First-Strand cDNA Synthesis Flexible Pack (NZYTech, Lisbon, Portugal) according to the manufacturer’s instructions, from 1 μg of total RNA from 3 biological replicates for each treatment and genotype.

Liver samples were homogenized using an Ultraturax homogenizer (D1000 handheld homogenizer; Benchmark Scientific, Inc., Sayreville, NJ, USA). The muscle tissues were homogenized by grinding them in liquid nitrogen in a cooled mortar and pestle. Total RNA was isolated using TRIzol reagent according to the Direct-zol RNA Miniprep (R2052; Zymo Research Orange, CA, USA) kit protocol. The quantity and purity of RNA were determined using a microplate reader (Synergy HT Multi-Mode Microplate Reader-SN 1712214, BioTek Instruments, Inc., Winooski, USA) as previously described^{24 (link)}. The quality and integrity of RNA were checked using a Qubit RNA IQ assay kit (# Q33222, Thermo Fisher Scientific) measured using a Qubit 4 fluorometer (Invitrogen, Thermo Fisher Scientific). The RNA IQ numbers ranged from 8.7 to 10. cDNA synthesis was performed using the LunaScript RT SuperMix Kit (New England Biolabs Inc. E3010L) with 200 ng total RNA and a PCR^max Alpha Thermal Cycler (Cole-Parmer Ltd. UK). The cDNA was stored at – 80 °C until the RT-PCR assay.

Total RNA was isolated from cultures of Y. enterocolitica strains grown in LBD medium at 26 °C (each culture in triplicate). After the cultures had reached OD₆₀₀ ~ 1, the cells were harvested by centrifugation, and RNA was isolated from the cell pellets using a Nucleo Spin RNA purification kit (Macherey-Nagel, Düren, Germany). RNA samples were treated with a TURBO DNA-free^TM kit (Invitrogen Waltham, MA, USA) to ensure the complete removal of contaminating DNA. The purity and quality of the RNA were assessed using a Qubit 4 fluorometer with a Qubit RNA IQ Assay Kit (Thermo Fisher Scientific). First-strand cDNA synthesis was performed using a Maxima H Minus First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed using 5x HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne, Tartu, Estonia) with a LightCycler 96 System (Roche, Basel, Switzerland). Oligonucleotide primers used for qPCR were purchased from Genomed S.A. (Warsaw, Poland) and are listed in Table S2. The levels of the amplified PCR products were normalized to that of a fragment amplified from the 16S rRNA reference gene. Fold changes were calculated using the Pfaffl method [110 (link)].

Total RNA was extracted using the miRNAeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA concentration was measured on a Qubit 4 Fluorometer (Thermo Fisher Scientific), using a Qubit RNA broad range assay kit (Thermo Fisher Scientific) and stored at -80°C. RNA integrity number (RIN) was measured using the Qubit RNA IQ assay kit (Thermo Fisher Scientific) and samples with values higher than 8 were considered for RNA sequencing (RNAseq) analysis. One µg of total RNA was sent for RNAseq analysis by Macrogen RNA-seq services (Seoul, South Korea) using the Truseq stranded total RNA Library with Ribo-Zero Gold (Illumina, San Diego, California, USA) and the NovaSeq 6000 (Illumina) of two independent biological replicates for each condition (control and treated with CM-1758) of 253 J, J82, RT112 and TCCSUP.