Quantifying Differential Gene Expression

Purified RNA samples (the same leaves as those used for the RNA-Seq analysis) were reverse-transcribed using the MLV-Reverse transcriptase (Takara Bio, Inc., Otsu, Japan). Thirteen genes from the DEGs list were used for RT-qPCR assay on a Bio-Rad CFX96 real-time PCR detection system. The detailed information of primer pairs was listed in Supplementary Table 1. The 2^−ΔΔCT method (Grabherr et al., 2011 (link)) was used to calculate the relative expression level of each gene.

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Liu J., Wang T., Weng Y., Liu B., Gao Q., Ji W., Wang Z., Wang Y, & Ma X. (2022). Identification and Characterization of Regulatory Pathways Controlling Dormancy Under Lower Temperature in Alfalfa (Medicago sativa L.). Frontiers in Plant Science, 13, 872839.

Publication 2022

MLV-Reverse transcriptase CFX96 real-time PCR detection system

Assay Expression gene Genes Primer Reverse transcriptase Rna seq

Corresponding Organization : China Agricultural University

Other organizations : Beijing Forestry University, National Animal Husbandry Service, Beijing Botanical Garden, Institute of Botany

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Variable analysis

independent variables

RNA samples from the same leaves used for RNA-Seq analysis

dependent variables

Relative expression level of 13 genes from the DEGs list

control variables

RNA samples were reverse-transcribed using the MLV-Reverse transcriptase (Takara Bio, Inc., Otsu, Japan)
RT-qPCR assay was performed on a Bio-Rad CFX96 real-time PCR detection system
The 2^(-ΔΔCt) method was used to calculate the relative expression level of each gene

controls

No positive or negative controls were explicitly mentioned in the provided information.

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