Experion rna analysis kit

For synovial organoids, lysis was performed using the TRI Reagent (Sigma-Aldrich); three synovial organoids were pooled together during the lysis step to yield sufficient RNA.
For synovial organoids, the aqueous phase was processed following lysis in the TRI Reagent for RNA extraction and purification. For cell culture, RNA was extracted using the Allprep RNA/Protein Kit (Qiagen). The quality and quantity of RNA were assessed using the Experion RNA Analysis Kit (Bio-Rad) and QuantIT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), respectively. Complementary DNA (cDNA) was synthesized using an iscript cDNA Synthesis Kit (Bio-Rad). Quantitative RT polymerase chain reaction (PCR) was performed using the CFX96 RealTime System (Bio-Rad) with LightCycler FastStart DNA Master plus SYBR Green I (Roche Diagnostics, Basel, Switzerland). The results were normalized to the housekeeping gene expression hypoxanthine-guanine phosphoribosyltransferase (HPRT). The sequences of the primers used in this study are available upon request.

Total RNA was extracted from retinas of treated animals after the indicated number of days using the RNeasy Plus mini kit (Qiagen). After treatment with RNase free DNase I (Ambion), the quality and quantity of RNA were analyzed using an Experion RNA Analysis Kit (Bio-Rad, Mississauga, Ontario). Fifty nanogram of total RNA was used for first-strand cDNA synthesis using the ProtoScript II reverse transcriptase (New England BioLabs). qPCR was performed using the GoTaq® qPCR Master (Promega, Madison, WI) on a CFX96™ real-time PCR detection system (Bio-Rad), as described previously (Taylor et al., 2010 (link)). Gene expression levels and PCR efficiency, along with its standard error, were calculated using the Bio-Rad CFX Manager, version 3.1 (Bio-Rad), which employs a proven comparative Cq (ΔCq) relative quantitation model with PCR efficiency correction and multiple reference gene normalization using GAPDH and Actin. Primer sequences for the specific and reference genes are listed in Table 2. Amplification efficiencies of primers were determined using serially diluted cDNA samples (Table 2). All PCR runs were performed in triplicate and the data analyzed by CFX Manager software (Bio-Rad).

A total of 16 CLL patients were included in this study. The CLL peripheral blood mononuclear cell (PBMC) samples were collected from the Section of Hematology and Coagulation, Sahlgrenska University Hospital. The CLL patients were diagnosed according to recently revised criteria [42 (link)] and samples were collected at the time of diagnosis. Clinical and molecular data are summarized in Additional file 2: data file 1A. All patients provided informed consent in accordance with the Helsinki Declaration and the study was approved by the local ethics review board. Genomic DNA and total RNA were extracted from CLL PBMCs and sorted B cell subpopulations with DNA (DNeasy Blood & Tissue Kit, 69504, Qiagen, Hilden, Germany) and RNA (miRNeasy mini kit, 217004, Qiagen, Hilden, Germany) extraction kits according to manufacturer’s protocol. The quality of RNA was measured using Experion RNA analysis kit (7007103, Bio-Rad, Hercules, USA). Five age-matched sorted CD+ 19 B cell DNA and RNA were bought commercially (3H Biomedical, Uppsala, Sweden). The quality of RNA was checked using 2100 Bioanlyser Instrument (Agilent, Santa Clara, United States) and the sent for RNA sequencing.

Total RNA was isolated on Days 0 (control, before differentiation) and 7 using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. Before starting RNA isolation, a stainless steel bead (5 mm, Qiagen) was added to a 2-mL tube (containing 1 mL Qiazol), and gels were disrupted for 2 min using a handheld rotor-stator homogenizer (TissueRuptor, Retsch, Germany). RNA was quantified using the NanoDrop ND-1000 instrument (Peqlab Biotechnologies, Wilmington, USA). We used the Experion RNA Analysis Kit (BioRad, Munich, Germany) to check the RNA quality. The Experion-automated electrophoresis system employs LabChip microfluidic technology to automate electrophoresis for RNA analysis and offers determination of total RNA and messenger RNA (mRNA) purity and concentration at nanogram levels. The RNA integrity number (RIN) is a numerical assessment of the integrity of RNA. A RIN over 8 indicates a high quality of RNA. In our test, we observed RIN numbers of 9.7–9.8.

Total RNA from hEEC and endometrial cell lines was isolated, after 2–4 days of culture, using the RNeasy Mini kit (Qiagen, The Netherlands). For the EMO, the RNeasy Micro kit (Qiagen, The Netherlands) was used. RNA was isolated according to the manufacturers’ guidelines. RNA concentration and quality were assessed via the Nanodrop method (Isogen Life Science, Belgium) and Experion RNA Analysis kit (Bio-Rad, Belgium). cDNA was generated from 1 µg of RNA using the First-Strand cDNA Synthesis Kit (GE Healthcare, Belgium). RT-qPCR was performed on triplicate cDNA samples using specific TaqMan gene expression assays (Supplementary Table I) (Life Technologies, Belgium) in the StepOne PCR system (Applied Biosystems, Belgium). Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) and Phosphoglycerate Kinase 1 (PGK1) were used as endogenous controls. Data is represented as mean ± SEM of 2^(−ΔCt) for which ΔCt = Ct_{gene of interest} − Ct_{geometric mean of HPRT1 and PGK1}.

To perform next-generation sequencing, aerial tissues derived from three biologically distinct 3-week-old A. tricolor plants were collected. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was examined via 1.2% (wt/vol) formaldehyde gel electrophoresis and with an Experion RNA analysis kit (Bio-Rad, Munich). Only high-quality RNA was used for next-generation sequencing performed on the Illumina HiSeq 4000 platform with 150 paired-end reads. For each dataset (AMR and AMG), 100 million reads were generated, and de novo assembly was performed with the Trinity tool. The assembled transcripts were annotated with BlastX in UniProt. Gene expression levels were normalized as FPKM values, and differentially expressed genes were identified according to an FDR < 0.05 and logFC > 2 or < −2 (Supplementary Tables S2, S3). An MA plot was generated based on the average concentration (logCPM) and fold-change (logFC) values to show the relative abundances of transcripts between AMR and AMG.