Lightcycler 480 syber green 1 master

Predesigned primer/probe sets for the 13 mitochondrial DNA (mtDNA) encoded subunits of the respiratory complexes I-V, Irs-1, Glut4 and Taqman Universal Mastermix were purchased from Applied Biosystems (supplementary Table 1). ABI 7700 Sequence Detection System (Applied Biosystems) was used for real-time PCR (RT-PCR) to quantify mRNA expression. As a control for between-sample variability, mRNA levels of metabolic and mitochondrial encoded genes were normalized to the geometric mean of the three housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (Gapdh), β-2 microglobulin (B2 m) and β-actin (Actb) (Vandesompele et al., 2002 (link)). The relative expression of the gene of interest was calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Results are reported as arbitrary units. Myosin heavy chain (MHC) gene expression was quantified using the LightCycler 480 SYBER Green I Master (Roche Applied Science, Indianapolis, IN). Normalization of MHC genes was performed using the geometric mean of the 3 reference genes: hypoxanthine phosphoribosyltransferase 1 (Hprt1), Cyclophilin A (Ppia), and Actb using the 2^−ΔΔCT method. Primer sequences and catalog numbers are provided in supplementary Tables 1, 2.

RNA was isolated using a Trizol reagent (Thermo Fischer Scientific, Vantaa, Finland). 400 ng of total RNA per sample was treated with RNase-free DNase I (Thermo Fischer Scientific, Vantaa, Finland). DNase I was inactivated with 50 mM EDTA at 65 °C for 10 min. The reverse transcription reaction was performed using random hexamer primers and RevertAid Reverse Transcriptase (Thermo Fischer Scientific, Vantaa, Finland). Complementary DNA (cDNA) was diluted 1:10 and stored at 20 °C until used for qPCR.
Quantitative PCR was performed using LightCycler 480 SYBER Green I Master (Roche, Espoo, Finland) and 100 μM primers, diluted 1:20, in 384-well plates with 10 μL total volume. All samples were analysed in triplicates or duplicates. Each reaction contained two negative controls (water and minus-reverse transcription). A combination of B-Actin, Ribosomal Protein S6, Beta-2-Microglobulin, and Gapdh were used as housekeepers for normalisation. Primer sequences are provided in Table 1.

The AcT and mAcT strain set to grow in normal condition and then AcT strain induced by adding 1 mM IPTG and placing both of the cultures under normal light conditions for 24 h. RNA was extracted from 15 ml of cell culture using Trizol reagents (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instruction. One microgram of total RNA was used as starting material for the cDNA generation using GoScript^TM Reverse Transcription System (Promega, United States) following the described instructions.
The qRT-PCR was performed applying Lightcycler480 Real-Time PCR system in a 20 μL reaction system containing 10 μL Light cycler^®480 SYBER^®Green I master (Roche) mix, 9 μL of template cDNA (100X diluted) and 0.5 μL of each PCR primer (5 picomoL concentration). Three biological along with three technical replications were performed for each sample. Data were analyzed using light cycler software. The data were normalized by using an internal controller, rnpB expression pattern. Fold change was calculated comparing between mutants. The significant change had been evaluated by student’s t-test applying the software GraphPad PRISM Version 5.01. The primers used for qRT-PCR analysis are also listed in Supplementary Table S2.

Total RNAs were extracted with TRIzol Reagent (Life Technologies), purified with RNeasy Mini Kit (QIAGEN) and treated with DNAseI (QIAGEN). cDNAs were prepared from 0.8 μg of RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and oligo-dT primers.–RT controls were included in qPCR reactions to discard genomic DNA contamination. qPCR was performed on Roche LightCycler 480 System (Roche) using LightCycler 480 SYBER Green I Master (Roche). Primers used are listed in S1 Table. Three independent biological replicates for each genotype were performed.