Iq5 rt pcr machine

Total RNA was isolated from whole seedlings and various tissues of mature plants using Trizol reagent (Invitrogen) and then treated with RNase-free DNase I (NEB) to remove genomic DNA contamination. First-strand cDNA was synthesized using RTace reverse transcriptase (Toyobo, Osaka, Japan) with oligo-dT primers. A 20-fold dilution of the resultant first-strand cDNA was subjected to PCR (22–35 reaction cycles) with gene-specific primers (Additional file 1). For the qRT-PCR reaction, first-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). A 10-fold dilution of the first-strand cDNA was subjected to qRT-PCR using FastStart Essential DNA Green Master (Roche, Basel, Switzerland) and an iQ5 RT-PCR machine (Bio-Rad, Hercules, CA, USA), in accordance with the manufacturers’ instructions. The PCR procedure was independently repeated at least three times. The relative gene expression levels are expressed as ratios of the abundance of the target gene’s mRNA to that of Act1 mRNA. Data were analyzed using the iQ5 2.1 software provided by the manufacturer. The gene-specific primers used for qRT-PCR are listed in Additional file 1.

Isolation of mRNA and cDNA preparation of wildtype strains, overexpression strains, deletion mutants and complementation M. smegmatis strains were performed and real-time PCR analysis was subsequently carried out according to previously described procedures [26] (link). The reactions were performed in a Bio-Rad IQ5 RT-PCR machine under the following thermocycling conditions: 95°C for 5 min and 40 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. Amplification specificity was assessed using melting curve analysis. Gene expression levels were normalized to the levels of sigA gene transcripts. An unrelated Ms6141 gene was used as a negative control. The degrees of expression change were calculated using the 2^−ΔΔCt method [26] (link). Average relative expression levels and standard deviations were determined from three independent experiments.

Total RNA from muscle and liver of eight individuals at the age of 110 days post hatch were extracted and reverse transcribed to single strand cDNA, following the instruction of the cDNA synthesis kit (Sigma, CA, United States). One primer pair (FoxK1-F and FoxK1-R) (Supplementary Table S1) was designed using Primer- blast software for qPCR. The qPCR was carried out in triplicates. The expressions of the foxK1 gene were examined by Quantitative real-time PCR (qRT-PCR) on an iQ5 RT-PCR machine (Bio-Rad, CA, United States). The elongation factor 1-alpha (EF1α) gene (see primers in Supplementary Table S1) was used as an internal control. The cDNA was amplified with the primers. PCR amplification was carried out in a total volume of 20 μl containing 1× Maxima^TM SYBR Green qPCR Master Mix (Fermentas, PA, United States), 0.25 μM of each primer and 10 ng template cDNA. The PCR program included a single cycle of 10 min at 95°C followed by 40 cycles of 15 s at 95°C, 30 s at 55°C, and 20 s at 72°C. To confirm the specificity of the amplification, after the completion of the qRT-PCR, a melting-curve analysis was conducted. The expression level of the foxK1 gene was analyzed using ΔΔCT method (Livak and Schmittgen, 2001 (link)).