Sv total rna isolation system kit

Wild type and Atwhy1why3 mutants were analysed by RNA-seq. Seed treatments were as follows: unaged seeds with 0 h imbibition, unaged seeds with 6 h imbibition, aged 7-day seeds with 6 h imbibition. The Promega SV Total RNA Isolation System kit (Madison, WI, U.S.A.) was used to extract total RNA according to the manufacturer's instructions [24 ]. Spectrophotometric quantification at 260 nm and samples between 1.7–2.1 A260/S280 ratio was accepted as sufficient purity (Nanodrop spectrophotometer). Total RNA samples were analysed by paired end reads using the Illumina NovaSeq 6000 Sequencing System (Novogene, Cambridge, U.K.). The paired and unpaired read data which was analysed using the Galaxy software (UseGalaxy.eu), produced by the Freiburg Galaxy Team [25 (link)]. Quality control was performed at each stage using FASTQC and MULTIQC. FASTQ files were filtered using trimomatic and aligned to the TAIR10 Arabidopsis genome using HISAT2. Paired and unpaired alignments were combined using samtools merge and featurecounts using to quantify gene fragments. Deseq2 was used to calculate normalised gene expression and identify differentially expressed genes (Supplementary Table S1), filtering for FDR values <0.01 and >2-fold changes in expression. Gene ontology enrichment was analysed using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost).

On day 5 of culture, total RNA was extracted using TRIzol LS reagent (Invitrogen). The extracted total RNA was purified using the SV Total RNA Isolation System kit (Promega, Madison, WI, USA). Then, it was quantified at different wavelengths (230, 260, 280, and 320 nm) in the GeneQuant 1300 device (GE Healthcare, Cardiff, WLS, UK) and assessed for its integrity using gel electrophoresis for the intact 28S and 18S ribosomal RNA (Figures S1 and S2). cDNA was synthesized from 1 µg of total RNA by reverse transcription using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). For real-time PCR, TaqMan probes (Applied Biosystems) (Table 1) were used in a CFX96 device (Bio-Rad, Hercules, CA, USA), with a final volume of 10 µL per reaction and 11.25 ng of cDNA. The results were analyzed based on the value of the cycle threshold (Ct), and the normalization and relative quantifications of gene expression were performed by the 2^−ΔΔCT method [25 (link)]. The data obtained were represented as the fold difference in the expression of the gene normalized to the constitutive gene (Gapdh), assigning a value of 1 to each marker in MC3T3-E1 cultures on the Control-Ti surface.

For real time qRT-PCR analysis, Bacillus sp. MB24 was inoculated into LB broth and incubated at 37 °C with agitation until the exponential phase. NaAsO₂ was added to the cultures to a final concentration of 0, 1, 10, 100, and 1000 μM, and the incubation was continued for another 30 min. Total RNA of Bacillus sp. MB24 was then extracted using the SV Total RNA Isolation System Kit (Promega) and further treated with DNase I (TaKara Bio Inc.). Real-time qRT-PCR was performed using AccessQuick RT-PCR System (Promega) with SYBR green1 (Bio-Rad, Hercules, CA, USA) along with the Corbett Rotor-Gene 3000 (Qiagen, Hilden, Germany) under the following conditions: reverse transcription at 45 °C for 45 min, followed by 25 amplification cycles, each of which consisted of denaturation for 30 s at 94 °C, annealing for 30 s at 50 °C, extension for 1 min at 72 °C, and finally detecting the fluorescence at 78 °C for 15 s. The primer sets RTarsR1-F/R, RTorf3F/R, and RTarsR2F/R were used to amplify the transcript of arsR1, orf3, and arsR2, respectively. The primer sequences are shown in Table S1. Primer sets 16F and 16R were used to amplify the bacterial 16S rRNA gene for normalization. Every reaction was repeated at least five times.

Total RNA was extracted from spleen tissue fragments using Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed by final purification with SV Total RNA Isolation System kit (Promega, Madison, WI, USA) in accordance with the manufacturer’s recommendation. RNA quantification was performed using Nanodrop spectrophotometer (NanoDrop Lite spectrophotometer; Thermo Fisher Scientific, Carlsbad, CA, USA). From 1 μg of RNA, the cDNA synthesis was performed using a High-Capacity cDNA Synthesis kit (Thermo Fisher Scientific, Carlsbad, CA, USA), following the manufacturer’s protocol. qPCR was performed using the SYBR^® Green PCR Master Mix (Applied Biosystems/ Thermo Fisher Scientific, Carlsbad, CA, USA) on a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Primers used to amplify cytokines (IFN-γ, TNF-α, IL-12, IL-10, TGF-β1) and iNOS genes were designed with the aid of Gene Runner version 6.0 using specific canine sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/ accessed on 18 November 2021) according to Menezes-Souza et al. [18 (link)]. GAPDH was used as reference for normalization of expression of the genes of interest. The results were expressed according to the 2^-ΔΔCt method.

They were determined using the SV Total RNA isolation system kit (Promega Corporation^®), following the manufacturer’s specifications. RNA was extracted from colonic tissue using the Trizol reagent (IntvitrogenTM, Waltham, MA, USA). The cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). For quantification by real-time PCR, the SYBR Green PCR Master Mix kit (Applied Biosystems) and primers (Forward-Reverse/Fw-Rv) were used, and mRNA expression was normalized using the housekeeping gene β-actin as internal control (Table 6). The mRNA relative quantification was calculated using the ΔΔCt method.

Total RNA from HepG2 cells was isolated using the SV Total RNA Isolation System kit (Promega Italia, Milano, Italy), following the manufacturer’s instructions. The reverse transcriptase (RT) reaction (20 μL) was carried out using 5 μg of total RNA, 100 ng of random hexamers and 200 units of SuperScript™ III RNase H-Reverse transcriptase (Life Technologies, Milano, Italy). Quantitative gene expression analysis was carried out on CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Segrate, Italy), using 18S rRNA for normalization. The primers used for real-time PCR analysis were listed in Table 1.

Total bacterial RNA was isolated using the SV Total RNA Isolation System Kit (Promega, Leiden, Netherlands) from 24 h cultures of L. rhamnosus strains incubated at 37°C with appropriate antibiotics and induced with nisin as previously described (De Keersmaecker et al., 2006a). To determine fluorescent protein gene expression, reverse transcription (RevertAid First Strand cDNA Synthesis Kit; Life Technologies Europe, Invitrogen, Gent, Belgium) with subsequent real‐time quantitative PCR (qRT‐PCR) (Power SYBR Green PCR Master Mix; Applied Biosystems Europe, Halle, Belgium) was performed. Primers were designed and synthetized by Integrated DNA Technologies (IDT) (Belgium) and are listed in Table S1. Relative abundance of mRNA corresponding to each fluorescent protein gene in relation to the housekeeping SrtA sortase mRNA was quantified after 40 amplification cycles of 15 s at 94°C and 1 min at 60°C. Expression plasmids containing each fluorescent protein‐encoding gene were used as cDNA plasmid controls, and mRNA from wild‐type L. rhamnosus GG and L. rhamnosus GR‐1 served as negative controls for each pair of primers.