Lightcycler 480 dna sybr green 1 master kit

For reverse transcription, the QIAGEN QuantiTect Reverse Transcription Kit (205311) was used according to the protocol with an input of 1.0 μg RNA per sample. Quantitative PCR was carried out in triplicate from three independent experiments, using the Roche Light Cycler 480 (SN: 1126) detection system and the Light Cycler 480 DNA SYBR Green I Master kit (Roche, 04707516001) and the primers listed in Supplementary Table S2. 36B4 and βcat served as reference genes. mRNA expression levels over time were calculated relative to day 0 which was set to 1. Statistical analyses were performed using a two-sided Student's t-test and the standard error of the mean was calculated for each dataset consisting of biological triplicates in technical triplicates each, individually.

Transcript levels of target genes were measured by quantitative real-time PCR (qPCR) using previously described methods (Ihry et al. 2012 (link)). RNA was isolated from appropriately staged animals using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 200 to 400 ng of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR was performed on a Roche 480 LightCycler using the LightCycler 480 DNA SYBR Green I Master kit (Roche). In all cases, samples were run simultaneously with three independent biological replicates for each target gene, and rp49 was used as the reference gene. To calculate changes in relative expression, the Relative Expression Software Tool was used (Pfaffl et al. 2002 (link)). For absolute quantification, the Ct (threshold cycle) for each target gene was compared to a standard curve generated for Ct’s of the respective amplicons at known concentrations.

RNA was isolated with TRIzol reagent as instructed (Invitrogen). For qPCR analysis, 1 μg of RNA was reverse transcribed (Applied Biosystems, Paisley, UK) using cycles of 25 °C (10 min), 37 °C (120 min), and 85 °C (5 min). cDNA templates were then used for qPCR using the LightCycler 480 DNA SYBR Green I Master kit (Roche Applied Science) in a LightCycler 480 II system (Roche Applied Science). All primers were from Sigma and were used at 0.5 μm. Actin was used as a control to normalize the data. Actin primers were 5′-ACTGGCATCGTGATGGACTC-3′ (forward) and 5′-TCAGGCAGCTCGTAGCTCTT-3′ (reverse). mTOR primers were 5′-TAAGAAAACGGGGACCACAG-3′ (forward) and 5′-TGAGAGAAGTCCCGACCAGT-3′ (reverse). Vps33A primers were 5′-CCCACAGGACTGCAGAAGA-3′ (forward) and 5′-TCCAACTGGGAGAGAAATCG-3′ (reverse).

RNA from tissues was quantified with the Nanodrop system and 400–500 ng aliquots were reverse-transcribed to cDNA using the Iscript Kit (Biorad, Hercules, CA). Quantitative PCRs were performed using the LightCycler 480 DNA SYBR Green I Master Kit (Roche Applied Science) and the LightCycler^® technology (LC480). The primer pairs HBS1-s (5′-TGGCCCTGGACATTGAGATT-3′) and HBS1-a (5′-TGGCTCCAGTCTTCACAGAG-3′), and K5-s (5′-TGGAGAGATGGCCCTGAAAG-3′) and K5-a (5′-CCACCTGTACTGAGACCTCC-3′) were used for the amplification of chicken HBS1 and K5, respectively. Quantities of target relative to reference transcripts were calculated according to a published mathematical model (Pfaffl 2001 (link)). The statistical significance of differences in expression levels was tested with ANOVA.
Other HBS keratin mRNAs were amplified by semiquantitative PCRs using the primers HBS2-s (5′-ACGCTGTCAGGAGAAGGAAC-3′), HBS2-a (5′-TTCTCACACAGCAAGCAGTC-3′), HBS3-s (5′-GAAGTCCCACCAGCAAGAGA-3′), and HBS3-a (5′-GTGGCGATCTCAATGTCCAG-3′), an annealing temperature of 60 °C and 32 amplification cycles. The ubiquitously expressed apoptotic protease, caspase-3, was amplified as a control gene using the primers CASP3-s (5′-TGGCGATGAAGGACTCTTCT-3′) and CASP3-a (5′-CTGGTCCACTGTCTGCTTCA-3′).

RNA isolation was performed with TRIzol reagent as instructed (Invitrogen). For qPCR analysis, 1ug RNA was reverse transcribed (Applied Biosystems, Paisley, UK) using cycles of 25°C (10 min), 37°C (120 min) and 85°C (5 min). cDNA templates were then used in qPCR with LightCycler 480 DNA SYBR Green I Master kit (Roche) in LightCycler 480 II system (Roche). All primers were from Sigma and used at 0.5uM. Actin was used as a control to normalize the data. Actin primers: 5′-ACTGGCATCGTGATGGACTC-3′ (forward) and 5′-TCAGGCAGCTCGTAGCTCTT-3′ (reverse); LC3-B primers: 5′-ACGATACAAGGGTGAGAAGCA-3′ (forward) and 5′-GTCCGTTCACCAACAGGAAG-3′ (reverse); p62 primers: 5′-AGATGAGGAAGATCGCCTTG-3′ (forward) and 5′-GGCATCTGTAGGGACTGGAG-3′ (reverse); mTOR primers: 5′-TAAGAAAACGGGGACCACAG-3′ (forward) and 5′-TGAGAGAAGTCCCGACCAGT-3′ (reverse); Vps34 primers: 5′-TCCTTGATGGTTGATGCAAA-3′ (forward) and 5′-CAGCAAAAAGAGCATGGACA-3′ (reverse).

Reverse transcription was performed using a QuantiTect Reverse Transcription Kit (Qiagen). All experiments were performed in two replicates with all statins (except for rosuvastatin and pravastatin, the least efficient statins from the microarray analyses). The RT-PCR was performed on LightCycler 2.0 System using a LightCycler 480 DNA SYBR Green I Master kit (Roche Diagnostics, Germany) and analyzed by LightCycler software. The resulting crossing point values were normalized using reference genes RPS9, TBP, and GAPDH. Relative fold changes of expression intensity in statin-treated against control samples were computed under the assumption of perfect effectivity of the PCR amplification. Statistical significance was estimated using Student’s t-test. All computations were performed within the R environment17 . The list of amplicons/primers of randomly selected target and housekeeping genes are provided in Suppl. Table 2.

RT4 cells were treated as indicated and RNA was isolated using TRIzol reagent following the manufacturer's instructions (Invitrogen). For qPCR analysis, 1ug RNA was reverse transcribed (Applied Biosystems, Paisley, UK) using the procedure of 25°C (10 min), 37°C (120 min) and 85°C (5 min). The resulting cDNA templates were subjected to qPCR using LightCycler 480 DNA SYBR Green I Master kit (Roche) with LightCycler 480 II system (Roche). GAPDH was used as a control to normalize the data. RIP1 primers (0.5 μM): 5′-AATAGTTCTCGTGTTCAGATTGGA-3′ (forward) and 5′-AGTGTTGGTTGGTGGTTGT-3′ (reverse) (Sigma); GAPDH primers (0.5 μM): 5′-ATCACTGCCACCCAGAAGAC-3′ (forward) and 5′-CAGTGAGCTTCCCGTTCAG-3′ (reverse) (Microsynth, Balgach, Switzerland).