DNA Sequencing Validation

To validate the transcription levels obtained from RNA-seq, real-time quantitative PCR (qPCR) was performed to compare gene expression patterns. The RNA extracts were used as template. Six representative genes of those identified by RNA-seq were tested (Table ​Table22). For cDNA synthesis, ImProm-II^TM Reverse Transcription System (Promega) was used. The qPCR experiments were performed using Corbett Rotor Gene 6000 (Corbett Life Science) with three technical replicates for each sample. Reactions were conducted in 25 μL reaction mixtures containing 1 μL of diluted cDNA, 2X Rotor-gene SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA), and gene-specific primers (Table ​Table22). The qPCR reactions included initial heating for 10 min at 95°C, followed by 40 cycles of 95°C, 10 s; 60°C, 15 s; 72°C 20 s. To calculate fold changes, the 2^-ΔΔCt method was used with the constitutively expressed 16S ribosomal RNA gene as a control for normalization.

Primer sequences used to validate the gene transcription levels obtained by RNA-seq approach.