Total RNA (1.5 μg) was used to generate cDNAs using the ThermoScript RT-PCR system to synthesize the first strand (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmoles of primers in a final volume of 25 μl of 2× concentrated RT2 Real-Time SYBR Green/ROX master mix (Qiagen) in an Applied Biosystems QuatStudio 6-Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50 °C for 2 min, one denaturation step at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 10s followed by annealing and elongation at 60 °C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Sequences of primers used for qPCR are provided in Table S1 .
Quatstudio 6 flex real time pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuatStudio 6-Flex Real-Time PCR instrument is a high-performance system designed for quantitative real-time PCR analysis. It features six independent thermal blocks for simultaneous sample processing and supports a wide range of reaction volumes.
Automatically generated - may contain errors
5 protocols using quatstudio 6 flex real time pcr instrument
1
Quantitative PCR of Total RNA Transcripts
2
RT-qPCR Analysis of mRNA Expression
3
Quantitative PCR of Gene Expression
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Total RNA (1.5 μg) was used to generate cDNAs using the ThermoScript RT‐PCR system to synthesize the first strand (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmoles of primers in a final volume of 25 μl of 2× concentrated RT2 Real‐Time SYBR Green/ROX master mix (Qiagen) in an Applied Biosystems QuatStudio 6‐Flex Real‐Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50°C for 2 min, one denaturation step at 95°C for 10 min and 40 cycles of denaturation at 95°C for 10s, followed by annealing and elongation at 60°C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Sequences of primers used for qPCR are provided in Table S1 .
4
RT-qPCR Analysis of mRNA Expression
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Analysis of specific mRNA by RT-qPCR was performed as described before (Shukla et al., 2018 (link)). Total RNA (1.5 μg) was used to generate cDNAs using the ThermoScript RT-PCR system for first-strand synthesis (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmol of primers, in a final volume of 25 μL of 2× concentrated RT2 Real-Time SYBR Green/R.O.X. master mix (Qiagen, Germantown, MD), using an Applied Biosystems QuatStudio 6-Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were 50°C for 2 min, one denaturation step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 10s, followed by annealing and elongation at 60°C. The relative gene expression of each transcript was normalized to GAPDH using the ΔΔCt method. Sequences of primers used for qPCR are provided in Table S1 .
5
Quantitative Gene Expression Analysis by RT-qPCR
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