qPCR Analysis of Alfalfa Transcripts

Total RNA from alfalfa seedlings was isolated using the Ultra-Pure RNA Kit (cwbiotech, Beijing, China) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using the ExScript RT kit (Takara Bio. Beijing, China). Quantitative PCR (qPCR) was performed on a Realplex 4 Master Cycler using a gene-specific primer (Table S1) using Super Real PreMix Plus with Syber Green 1 (TIANGEN Biotech., Beijing, China). Relative gene expression was assessed using a comparative cycle threshold method [40 (link)]. The relative gene expression was calculated as follows: 2^−ΔΔCT (ΔCT = CT, gene of interest^-CT versus actin) as described by [41 (link)], where the actin gene was used as a reference. A mixture cDNA of 30 leaf samples was used to perform the qPCR reactions to determine the primer efficiency as described previously [42 (link)]. All primer amplification efficiencies were between 99% and 104% (Table S1). Three complete biological and technical replicates were performed.