Detailed protocols are provided in our previous report.6 (link) RT-qPCRs were run on a qTOWER 2.2 Real-Time PCR system (Analytik Jena, Jena, Germany) using reaction mixtures containing 1 μL of diluted cDNA and 5 μL of 2× SYBR® Green Supermix (DBI, Ludwigshafen, Germany). The thermal cycling conditions were as follows: 95 °C for 3 min followed by 39 cycles of 95 °C for 10s and 60 °C for 30s. Relative eRNA levels in PBMCs were calculated using the following equation: amount of target = 2−ΔCt, where delta Ct = Ct eRNAs – Ct GAPDH. The sequences of the eRNA-specific primers used are provided in Supplementary File, Table S2
Protocol detail
Quantifying eRNA Levels in PBMCs
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Biological
Cdna
Gapdh
Green 5
Primers
Replicate
Sybr green
Corresponding Organization :
Other organizations : Wenzhou Medical University, Zhejiang University, First Affiliated Hospital Zhejiang University, First Affiliated Hospital of Wenzhou Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- Relative eRNA levels in PBMCs
- Thermal cycling conditions: 95 °C for 3 min followed by 39 cycles of 95 °C for 10s and 60 °C for 30s
- RT-qPCR reaction mixture: 1 μL of diluted cDNA and 5 μL of 2× SYBR® Green Supermix
- RT-qPCR system: qTOWER 2.2 Real-Time PCR system (Analytik Jena, Jena, Germany)
- Use of GAPDH as a reference gene for relative quantification
- Positive control: None mentioned
- Negative control: None mentioned
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