Faststart universal sybr green pcr master mix

Total-RNA from cells was extracted using Trizol reagent (1596-026) from Invitrogen Life Technologies, reverse transcription was conducted using RevertAid^TM Reverse Transcriptase (EP0441) from Thermo Fisher Scientific Inc., and quantitative PCR was conducted using FastStart Universal SYBR Green PCR Master mix (4913914001) from Roche, according to manufacturer’s procedure. Forward (F) and reverse (R) primers were used as follows: FoxO1, F: 5′-TGGACATGCTCAGCAGACATC-3′ and R: 5′-TTGGGTCAGGCGGTTCA-3′; NF-κB1, F: 5′-CCTGGATGACTCTTGGGAAA-3′ and R: 5′-TCAGCCAGCTGTTTCATGTC-3′, GAPDH, F, 5′-ATGGGGAAGGTGAAGGTCG-3′ and R, 5′-GGGGTCAT TGATGGCAACAATA-3′, and synthesized by Invitrogen29 (link)30 (link). TaqMan microRNA assay for miR-9 was purchased from Applied Biosystems Inc., and U6 small nuclear RNA (U6 snRNA) was used as normalization control. All real-time amplifications were measured in triplicates and performed with the ABI Prism 7300 sequence detection system (Applied Biosystems) as we previously described28 (link)31 (link). The fold-change of miR-9, FoxO1, and NF-κB1 was calculated using the 2^−ΔΔCT method.

The total RNA was extracted using Trizol reagent (Invitrogen Corp, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted RNA was reverse transcribed into cDNA, and the cDNA was used as a template. qPCR was performed on the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using FastStart Universal SYBR Green PCR Master Mix (Roche, Basel, Switzerland). GAPDH was used as an internal control. All data were measured in triplicates and calculated using the 2^−∆∆CT method. The primers for human CCL2 were forward: 5′-CAGCCAGATGCAATCAATGCC-3′ and reverse: 5′-GGAATCCTGAACCCACTTCT-3′.

A total of 500,000 cells were seeded per well in 6–well microplates (Falcon Mutliwell), 24 h prior to any treatment. Cells were then treated with indicated drug and time. TRIzol (Invitrogen) was used to extract RNA. One microgram of RNA was used for reverse transcription (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). cDNAs were diluted five times before being used as described by the provider (4 μL/reaction) with FastStart Universal SYBR Green PCR Master Mix or FastStart Universal Probe Master Mix TaqMan (Roche, 30, cours de l’Ile Seguin 92650 Boulogne-Billancourt Cedex, France) for qPCR with 20 μL as the total volume for each reaction. For miRNA analysis, we used RT miScript mix, Hi Spec reagent, (Qiagen, 3 avenue du Canada LP 809 91974 Courtaboeuf Cedex; France) and miScript SYBR Green PCR Kit (Qiagen) as described by the provider. qPCRs were performed with 7500 Real Time PCR System (Applied Biosystems, Boulevard Sébastien Brant - F67403 Illkirch Cedex, France). Relative expression levels were normalized to TBP, G3PDH or RNU6 using the 2^(−ΔΔCt) method [51 (link)]. Primers used are provided in Supplementary Figure S9.

RT-qPCR was performed using the LightCycler® 480 Real-Time PCR System (Roche). Reactions of 25 µl were comprised of 12.5 µl of FastStart Universal SYBR Green PCR Master Mix (Roche, Germany, 2X conc.), 1 µl of each forward and reverse primer (10 µM conc., 0.4  µM final conc.), 5.5 µl of PCR grade water, and 5 µl of cDNA (generally at ~150–170 ng/µl dilutions). The cycling conditions were denaturation at 95 °C for 5 min, followed by 50 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 10 s. Melting curve analysis was performed after each cycle to ensure primer specificity. All samples were amplified in technical triplicates and a mean value was calculated. Four (M. cerebralis) to five (S. molnari) biological replicates were used for each sample, with the exception of C. shasta (only 3 replicates available). qPCR efficiency was predicted for each gene based on the slope of a linear regression model^{46 (link)} using a series of 5-fold dilutions (1:5, 1:25, 1:125, 1:625). Standard curves were built using Roche Light Cycler 480 Software version 1.5.0 SP4. Generally, for best amplification results efficiency ranges of 90–110% and standard curve slopes of −3.58 to −3.10 were considered optimal^{47 (link)}.

Total RNA was extracted using the TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. The extracted RNA was reverse transcribed into cDNA, and the cDNA was used as a template to perform quantitative real-time PCR on the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using FastStart Universal SYBR Green PCR Master Mix (Roche, Basel, Switzerland). GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control.
qPCR was performed using the following conditions: 94 °C for 3 min for 1 cycle; 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, for 40 cycles. The sequences of the primers used in the experiment were: VPS4A-up: 5′-GTGATGGAGAAGCCCAACATAC-3′; VPS4A-low: 5′-CAAGTGTGGGAATTTGATTGGC-3′; VPS4B-up: 5′-CGACCAAATGTGAAATGGAGTGA-3′; VPS4B-low: 5′-TCCAGGCGGCCCAAATAATAG-3′.
After determining the specificity of amplification by melting curve analysis, the relative expression of the target mRNA was calculate using the 2^−ΔΔCT method.