Turbo dna free kit

HCT116 cell cultures were treated with 5-FU (20 μM) or oxaliplatin (12.5 μM) for 48 h (n = 4/group). After treatment, cultures were processed for RNA extraction with Tri-reagent (TR 118, Medical Research Centre) according to the manufacturer’s instructions. Genomic DNA was removed using the Ambion Turbo DNA-free kit, followed by reverse transcription using an M-MLV reverse transcriptase for 2 h at 37 °C (M1705, Promega). Quantitative real time PCR was carried out in technical triplicates on a Light Cycler 480 (Roche) using the SYBR select master mix (4472918, Life Technologies) according to manufacturer’s instructions. Reactions for each target were optimised for an efficiency >1.8 and specificity was confirmed via melt-curve analysis. The following primers (KiCqStart SYBR green, Sigma) were used: translationally controlled tumour protein (TCTP/TPT1) (sense, 5’-TACTCTTTCTGGTCTCTGTTC-3’; antisense, 5’-CAAGTTTCACAAAAGAAGCC-3’), and β2-tubulin (B2T) (sense, 5’-AAGGACTGGTCTTTCTATCTC-3’; antisense, 5’-GATCCCACTTAACTATCTTGG-3’) at 400 nM in 20 μL reactions with 80 ng cDNA. Treatment effects on TCTP mRNA expression were assessed using one-way ANOVA, followed by Tukeys post-hoc analysis, where applicable. Analyses were performed using SPSS 19.0 (IBM). Statistical significance was accepted at P < 0.05 and data are presented as the mean ± standard error of the mean (SEM).

Total RNA was isolated from shoots and roots representing each time point using the ZR Plant RNA MiniPrep™ Kit (Zymo Research, Irvine, CA, USA). The quality and quantity of isolated RNAs were checked by agarose gel electrophoresis and spectrophotometrically using a BioPhotometer (Eppendorf BioPhotometer plus, Hamburg, Germany). Residual DNA was eliminated using a TURBO DNA-free™ Kit (Promega, Madison, WI, USA).

Wild-type strain XHG3 and the ΔphoP mutant were cultured in YEB medium at 28 °C to OD₆₀₀ = 1.0, with two biological repeats performed for each strain. Total bacterial RNA was extracted using the SV Total RNA Isolation System (Promega), with eluted RNA then DNase-treated using a Turbo DNA-free Kit (Promega) as per the manufacturer’s instructions. The quality and quantity of the total RNA was assessed using agarose gel electrophoresis, spectrophotometry, and an Agilent 2100 Bioanalyzer. Transcriptome library construction and sequencing were performed by Novogene (Beijing, China). Clean read data were obtained following sequencing and data filtering, and resulting reads were mapped to the X. citri subsp. citri strain 306 reference genome. Gene expression, as indicated by the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), was calculated using HTSeq, with difference in expression between strains ΔphoP and XHG3 determined using DESeq.
Differentially-expressed genes (DEGs) were designated based on q-values < 0.005 and a minimum absolute |log₂(Fold Change)| > 1, and were exported as a tabular file (Supplementary Table S2). DEGs were then classified and enriched based on gene ontology (GO) database (http://www.geneontology.org/ and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.