Rnaase free dna set

Result obtained in the RNA-seq analysis were validated by quantitative reverse transcription PCR (qRT-PCR) of 20 selected genes, which represented differentially and non-differentially expressed genes in the presence of apigenin and salt. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche) and RNAase Free DNA Set (Qiagen), according to the manufacturer’s instructions. This (DNA-free) RNA was reverse transcribed into cDNA using a QuantiTec Reverse Transcription Kit (Qiagen). Quantitative PCR was performed using a LightCycler 480 (Roche) with the following conditions: 95 °C, 10 min; 95 °C, 30 s; 50 °C, 30 s; 72 °C, 20 s; forty cycles, followed by the melting curve profile from 60 to 95 °C to verify the specificity of the reaction. The R. tropici CIAT 899 16S rRNA gene was used as an internal control to normalize gene expression. The fold-changes of two biological samples with three technical replicates of each condition were obtained using the ∆∆C_t method [38 (link)]. Selected genes and primers are listed in Additional file 2.

Results obtained in the RNA-seq analysis were validated by quantitative reverse transcription PCR (qRT-PCR) of 13 selected nodulation genes which represented differentially and non-differentially expressed genes in the presence of apigenin and salt. Total RNA was isolated using a High Pure RNA Isolation Kit (Roche) and RNAase Free DNA Set (Qiagen), according to the manufacturer’s instructions. This (DNA-free) RNA was reverse transcribed into cDNA using a QuantiTec Reverse Transcription Kit (Qiagen). Quantitative PCR was performed using a LightCycler 480 (Roche) with the following conditions: 95 °C, 10 min; 95 °C, 30 s; 50 °C, 30 s; 72 °C, 20 s; forty cycles, followed by the melting curve profile from 60 to 95 °C to verify the specificity of the reaction. The R. tropici CIAT 899 16S rRNA gene was used as an internal control to normalize gene expression. The fold-changes of two biological samples with three technical replicates of each condition were obtained using the ΔΔCt method41 (link). Selected genes and primers are listed in S5.

RNA extraction, cDNA synthesis and quantitative RT-PCR (qPCR) experiments were performed as previously described (Pérez-Montaño et al., 2016 (link)). Briefly, total RNA was isolated from bacterial cultures grown in TY medium using a High Pure RNA Isolation Kit (Roche) and RNAase Free DNA Set (Qiagen, USA), according to the manufacturer’s instructions. Verification of the amount and quality of RNA samples was carried out using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, USA) and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Two independent total RNA extractions were obtained for each condition.
This (DNA-free) RNA was reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative PCR was performed using a LightCycler 480 (Roche) with the following conditions: 95 °C, 10 min; 95 °C, 30 s; 55 °C, 30 s; 72 °C, 20 s; 40 cycles, followed by the melting curve profile from 60 to 95 °C to verify the specificity of the reaction. The R. tropici CIAT 899 16S rRNA gene was used as an internal control to normalize gene expression. The fold-changes of two biological samples with three technical replicates of each condition were obtained using the ΔΔC_t method (Pfaffl, 2001 (link)). Selected genes and primers are listed in Supplementary Table S2.