RNA from the sorted cells was extracted with the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific, Lithuania) according to the manufacturer recommendations. The RNA was quantified in a Nanodrop spectrophotometer and 10 ng of total RNA was used to amplify the mRNA transcripts using real-time qPCR with the SYBR Green RT-PCR one-step kit (Agilent, USA). The amplification protocol was 50 °C for 30 min and 35 cycles of 94 °C for 30 min and 55 °C for 1 min. The amplification was carried out using the primers previously described (Maisonnasse et al., 2016a (link)). The quantification was performed using the Ct values and the formula 2−ΔCt and for each animal, the expression is presented as the relative expression to the gene with the highest expression as previously described (Maisonnasse et al., 2016a (link); Silva-Campa et al., 2010 (link)).
Sybr green rt pcr one step kit
Manufactured by Agilent Technologies
Sourced in United States
The SYBR Green RT-PCR one-step kit is a reagent system designed for reverse transcription and real-time PCR amplification of RNA samples. The kit includes all necessary components for both steps in a single reaction mixture, allowing for simplified workflow and reduced handling time.
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2 protocols using sybr green rt pcr one step kit
1
Quantifying mRNA Transcripts via qPCR
2
Gene Expression Analysis of Sorted Cells
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RNA from sorted or cultured cells was extracted with the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s recommendations. Ten nanograms of total RNA was used to amplify the mRNA transcripts using real-time PCR with the SYBR Green RT-PCR One-Step Kit (Agilent, Madison, WI, USA). The amplification protocol was as follows: 50 °C for 30 min and 35 cycles of 94 °C for 30 min and 55 °C for 1 min. The primers used to amplify FLT3, IL-4, TGFB, FoxP3, IL-10, IFN-γ, IFN-α have been previously described by others and us. The results were analyzed with the formula 2−ΔΔCt and are expressed as relative ratios.
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