For the analysis of mRNA levels, cells were grown in 6-well culture dishes for 24 h in complete medium at 37°C in 5% CO2. Cells were lysed, and total RNA was extracted using the InviTrap Spin Universal RNA Mini kit (Stratec Molecular). cDNA was obtained using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen), all according to the manufacturer’s instructions. The Q-RT-PCR was performed in internal triplicates with the specific and efficient (efficiency between 90 and 110%) primer pairs (Sigma-Aldrich) listed in Table S2. The Q-RT-PCR reaction was performed using the SYBR green detection reagent (Applied Biosystems) in StepOne Real-Time PCR System machines (Applied Biosystems) using the StepOne software v2.3, all according to the manufacturer’s instructions with standard Q-RT-PCR cycling conditions. For each biological condition, the fold change (2−ΔΔCT) of target mRNA was normalized to that of GAPDH.
Stepone real time pcr system machines
Manufactured by Thermo Fisher Scientific
The StepOne Real-Time PCR System is a compact, easy-to-use instrument designed for real-time PCR analysis. It features a 96-well block format and can perform a variety of real-time PCR applications, including gene expression analysis, genotyping, and pathogen detection.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green detection reagent, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (3 mentions)
Rnaeasy kit, by Qiagen (2 mentions)
Stepone software v2, by Thermo Fisher Scientific (2 mentions)
Primer pair, by Merck Group (1 mentions)
Invitrap spin universal rna mini kit, by Stratec (1 mentions)
3 protocols using stepone real time pcr system machines
1
Quantitative RT-PCR Analysis of mRNA Levels
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using RNAeasy kit (Qiagen). 500 ng total RNA was subjected to reverse transcription using SuperScript III First-Strand Synthesis Supermix (Invitrogen) according to the manufacturer’s instructions. cDNAs obtained this way were diluted 1/10 and used for PCR. The RT-qPCR reaction was performed using the SYBR green detection reagent (Applied Biosystems) in StepOne Real-Time PCR System machines using the StepOne software v2.3 (Applied Biosystems). Primer sequences are described in the supplementary information. Changes in gene expression were calculated with the 2−ΔΔCT method (Livak and Schmittgen 2001 (link)) using GAPDH as the housekeeping gene.
3
Real-Time PCR Gene Expression Analysis
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Total RNA was extracted using RNAeasy kit (Qiagen). 500 ng of total RNA was subjected to reverse transcription using SuperScript III First-Strand Synthesis Supermix (Invitrogen) according to the manufacturer´s instructions. cDNAs obtained this way were diluted 1/10 and used for PCR. The RT-qPCR reaction was performed using the SYBR green detection reagent (Applied Biosystems) in StepOne Real-Time PCR System machines using the StepOne software v2.3 (Applied Biosystems). Primer sequences are described in the supplementary information. Changes in gene expression were calculated with the 2 -ΔΔCT method (Livak and Schmittgen 2001) (link) using GAPDH as the housekeeping gene.
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