Quantifying Gene Expression via qRT-PCR

To analyze gene expression levels, qRT-PCR was carried out with a 2 × SYBR Green qPCR Mix Kit (Aidlab, China) in a 25 μl volume on a 7500 Fast Real-Time instrument (Thermo Fisher, Waltham, MA, United States) using the following cycling protocol: 3 min at 94°C followed by 40 cycles of 20 s at 94°C, 20 s at 55°C, and 30 s at 72°C (signal acquisition at 72°C). ZjActin was used as a reference gene (Bu et al., 2016 (link)). The 2^–ΔΔCt method was used to calculate the relative gene expression levels (Livak and Schmittgen, 2001 (link)).

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Li M., Hou L., Zhang C., Yang W., Liu X., Zhao H., Pang X, & Li Y. (2022). Genome-Wide Identification of Direct Targets of ZjVND7 Reveals the Putative Roles of Whole-Genome Duplication in Sour Jujube in Regulating Xylem Vessel Differentiation and Drought Tolerance. Frontiers in Plant Science, 13, 829765.

Publication 2022

2 × SYBR Green qPCR Mix Kit 7500 Fast Real-Time instrument

Gene Gene expression Sybr green

Corresponding Organization : Beijing Forestry University

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Variable analysis

independent variables

QRT-PCR cycling protocol (3 min at 94°C, 40 cycles of 20 s at 94°C, 20 s at 55°C, and 30 s at 72°C)

dependent variables

Gene expression levels

control variables

Reference gene: ZjActin
SYBR Green qPCR Mix Kit (Aidlab, China)
7500 Fast Real-Time instrument (Thermo Fisher, Waltham, MA, United States)

controls

Positive control: Not specified
Negative control: Not specified

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