Faststart dna master sybr green

Total RNA was extracted using TRIzol reagent (#15596018, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A reverse transcription kit (#04379012001, Roche, Mannheim, Germany) was used to transcribe the cDNA. Real-time qPCR was performed with cDNA as a template using a Light Cycler system with FastStart DNA Master SYBR Green (#06402712001, Roche, Mannheim, Germany). The 36B4 gene was used as an internal control, and the primer sequences used in the present study are listed in Table A1.

ChIP samples were resuspended in 1 ml. 5 μl of ChIP material was combined with 0.5 μM of primers and FastStart DNA Master SYBR Green (Roche, 03003230001) in a 15 μl reaction and amplified in a LightCycler (Roche). Samples were normalised for input and compared against a negative control point (β-actin promoter). Results and error bars are the mean average and standard deviations of three independent experiments. P-values were calculated using Student’s T-test. See Supplementary Table 1 for a full list of primers used in this study.

Total RNAs (2 µg) were reverse-transcribed with Moloney murine leukemia virus RT (Invitrogen) using random hexanucleotides. Real-time quantitative PCR was done using the “FastStart DNA Master SYBR Green” kit and the Lightcycler apparatus (Roche Diagnostic). The mRNA levels of interest were normalized to the 18S RNA amount, which was not affected by the different fasting periods (in the liver) and the pyruvate treatment (in the hepatocytes).

Total RNA of muscle and heart was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The cDNA was synthesized using the SuperScript III First-Strand Synthesis System for real-time polymerase chain reaction (RT-PCR) (Life Technologies, Carlsbad, CA, USA). RT-PCR was performed on a LightCycler using the FastStart DNA Master SYBR Green (Roche Diagnostics, Indianapolis, IN, USA) according to the protocol provided by the manufacturer. The expression level of 18S ribosomal RNA was used as a control for qPCR. Sequences of primers used for RT-PCR were as follows: SIRT3-F 5′-CGGCTCTATACACAGAACATCGA-3′ and SIRT3-R 5′-GTGGGCTTCAACCAGCTTTG-3′; atrogin-1-F 5′-GCAAACACTGCCACATTCTCTC-3′ and atrogin-1-R 5′-CTTGAGGGGAAAGTGAGACG-3′; MuRF-1-F 5′-ACCTGCTGGTGGAAAACATC-3′ and MuRF-1-R 5′-CTTCGTCCTTGCACATC-3′; 18S ribosomal RNA-F 5′-AACGAGACTCTGGCATGCTAACTAG-3′ and 18S ribosomal RNA-R 5′-CGCCACTTGTCCCTCTAAGAA-3′.

DNA was extracted from pools of eight whole eyes and eluted in 200μl of elution buffer, as previously described [19 (link)]. Total DNA concentration was determined by Nanodrop^®. Toxoplasma specific DNA was quantified with real-time PCR on a LightCycler (Roche Diagnostics) using the Faststart DNA Master SYBR Green (Roche Diagnostics, Meylan, France), in 5 μl of DNA solution and a reaction volume of 20 μL, following the manufacturer’s recommendations. The repetitive TgB1 gene of T. gondii was chosen as target, as it allows sensitive quantification even for avirulent strains [20 (link)]. The following primers were used: 5’-GGA GGA CTG GCA ACC TGG TGT CG CG-3’ and 5’-TTG TTT CAC CCG GAC CGT TTA GCA G-3’. The parasite load was calculated using external standards, extracted from known numbers of parasites, run in parallel, and calculated as parasites per μg DNA. The standard curve showed high degrees of linearity at least between 10¹ and 10⁶ parasites per reaction. Viability of the parasites was assured for each batch by infecting cell cultures.

Total RNA was extracted with TRIzol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. To examine the expression, qPCR was performed by the LightCycler 480 apparatus (Roche) according to the manufacturer's instructions using FastStart DNA Master SYBR Green (Roche). β-actin was used as an endogenous control. Double-stranded DNA specific expression was tested by the comparative Ct method using 2^−ΔΔCt[39] (link). Primers were designed using Primer3 software (http://frodo.wi.mit.edu/). The primer sets were as follows: Fut8: 5′-ACCAAGAAGCTTGGCTTCAA-3′ (forward), and 5′-TTTGTCCACTTGCATTCTGC-3′ (reverse); β-actin: 5′- GGACTTCGAGCAAGAGATGG-3′ (forward), and 5′-AGCACTGTGTTGGCGTACAG-3′.