Innuprep rna kit

RNA was isolated using InnuPrep RNA kit (Analytik Jena, Jena, Germany) and cDNA was generated using SuperScript IV cDNA Synthesis Kit (ThermoFisher Scientific). For qPCR, Eva Green Dye (Solis Biodyne, Tartu, Estonia) was used and respective primer pairs; p50 (fw: 5′-CACTTAGCAATCATCCACCTT-3′, rev: 5′-AGCCCTCAGCAAATCCT-3′), p65 (Qiagen; QT02324308), c-Rel (Qiagen; QT00052472), p52 (fw: 5′-GGGGCATCAAACCTGAAGATTTCT-3′, rev: 5′-TCCGGAACACAATGGCATACTGT-3′), RelB (Qiagen QT00038640), CTGF (fw: 5′-CTCGCGGCTTACCGACTG-3′, rev: 5′-GGCTCTGCTTCTCTAGCCTG-3′), PAI-1 (SERPINE-1) (fw: 5′-CTCTCTCTGCCCTCACCAAC-3′, rev: 5′-GTGGAGAGGCTCTTGGTCTG-3′) αSMA (fw: 5′-CGTGGGTGACGAAGCACAG-3′, rev: 5′-GGTGGGATGCTCTTCAGGG-3′), collagen IAI (fw: 5′-GCTCCTGCTCCTCTTAGCG-3′, rev 5′-CCGTTCTGTACGCAGGTGAT-3′), RNA-polymerase IIA (fw: 5′-GGAGATTGAGTCCAAGTTCA-3′, rev: 5′-GCAGACACACCAGCATAGT-3′), integrin αv (fw: 5′-CACTTCGGCGATGGCTTTTC-3′, rev: 5′-GTAGCAGGAGTCCCGAGAGA-3′), integrin α2 (fw: 5′-GTGGCTTTCCTGAGAACCGA-3′, rev: 5′-GATCAAGCCGAGGCTCATGT-3′), integrin β1 (fw: 5′-ACGCCGCGCGGAAAAGATGA-3′, rev: 5′-GCACCACCCACAATTTGGCCC-3′), Data analysis was performed using QuantStudio5 and QuantStudio Design and Analysis Software (ThermoFisher Scientific).

Total RNA extraction was carried out with innuPREP RNA Kit (Analytik Jena, Jena, Germany). One microgram of total RNA was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time PCR (qRT-PCR) quantification was performed using gene-specific oligonucleotides (see Supplementary Table S1 at JXB online) and SensiMix™ SYBR Low-ROX (Bioline, Luckenwalde, Germany) in duplicate assays (7500 real-time PCR system, Applied Biosystems/Thermo Fisher Scientific, Dreieich, Germany). UBQ5 and S16 were used to normalize expression values (Vandesompele et al., 2002 (link)) as normalized relative quantities (NRQ). Brassica napus UP1 and UBQ9 were used as reference genes for canola samples (Chen et al., 2010 (link)). Cycle values and efficiency of reaction were extracted from the raw data with the qPCR package (Spiess, 2018 ) and NRQs were calculated by Microsoft Excel (Hellemans et al., 2007 (link)).

RNA was isolated using the InnuPrep RNA Kit (Analytik Jena) 24 h after treatment with TGFβ, EGF, or a combination of both and reverse transcribed with M-MLV reverse transcriptase (Thermo Fisher Scientific). SYBR green-based qPCR was performed on a CFX96 Thermocycler (BioRad) using iTaq universal SYBR green super mix (BioRad). Primer pairs are listed in Supplementary Table S1. Changes in gene expression are shown as log2 of 2^–ΔΔCt compared to the respective untreated control and normalized to GAPDH which was used as reference gene.

Total RNA was isolated from tumor cells or zebrafish larvae using the InnuPrep RNA Kit (Analytik Jena, Jena, Germany) and reverse transcribed with M‐MLV reverse transcriptase (Thermo Fisher Scientific). Quantitative real‐time PCR was performed on a CFX96 Thermocycler (BioRad, Hercules, CA, USA) using iTaq Universal Probes or SYBR Green Supermix (BioRad). Taqman probe IDs and primer sequences are listed in Table S1. Changes in gene expression are shown as log2 of 2−ΔΔCt compared to the respective untreated control and are normalized to a reference gene as indicated.

Total RNA was extracted from 100 mg of fresh, fully expanded young leaf samples (taken on 14 DAO) using an innuPrep RNA kit (Analytik Jena, Jena, Germany). After treating the samples with DNase I (Sigma), the extracted RNA was reverse transcribed into cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The cDNAs were then used as templates for the qPCR reaction using specifically designed primers provided in Table 4. Real-time PCR reactions were performed using (CFX96, BioRad Laboratories, Hercules, CA, USA) and the assay contained 5 µL of Sso Advanced™ Universal SYB^® Green superMix (Bio-Rad, Hercules, CA, USA), 2µL of forward and reverse primers, 1 µL of template cDNA and 1 µL of PCR water in a total volume of 10 µL. The thermal cycle’s set up was as follows: 98 °C for 1 min; 44 cycles of 97 °C for 15 s, 58.5 °C for 20 s, and 72 °C for 35 s. The expression levels of antioxidant enzymes were analyzed by the comparative Ct method.

For RNA isolation, antibody-labeled cells were sorted directly into RLT+lysis buffer and purified according to manufacturer’s protocol (RNEasy mini Kit, Quiagen). RNA from whole tissues was isolated with mechanical tissue dissemination using ceramic beads followed by column-based RNA isolation (Innuprep RNA Kit, Analytik Jena). RNA was reversely transcribed into cDNA according to manufacturer’s protocol (Reverse Transcriptase II, life technologies). cDNA was used with Taqman probes and Taqman master mix or with Sybr primers and Sybr master mix in a Viia7 real-time PCR system (all ThermoFisher). Gene expression was normalized to housekeeping gene expression (Hprt) with the formula: relative gene expression = 2^{−(Ct (gene X)—Ct (Hprt))}. Primer sequences and designations are listed in Supplementary Table 1.