Chitinase Gene Expression in T. pseudonana

After 48 h of stress treatment, a 1 mL sample was taken from each flask for measurement of chitinase activity using the Solarbio Chitinase Assay Kit (Cat#BC0820) following the manufacturer’s instructions. The remaining 49 mL of T. pseudonana cells in each flask were collected by centrifugation at 2850 g, washed once with fresh medium, and flash-frozen in liquid nitrogen. The T. pseudonana cell pellets were homogenized by vortexing in TRIzol reagent (Invitrogen, Waltham, MA, USA), then centrifuged at 9700 g. The supernatants were used for RNA extraction with chloroform and isopropanol. RNA pellets were resuspended in diethyl pyrocarbonate-treated water, followed by elimination of genomic DNA and synthesis of complementary DNA (cDNA) with a PrimeScript RT reagent kit (Takara, Japan). The synthesized cDNA was then used for qRT–PCR experiments.
The transcriptional profiles of 14 randomly selected T. pseudonana chitinase genes were obtained by qRT–PCR using TB Green™ Premix Ex Taq™ II (Takara, Japan) and a Thermal Cycle Dice™ Real Time System (Takara, Japan). Gene-specific qRT–PCR primers were designed using the NCBI Primer-BLAST website [70 (link)]. Information on primers is provided in Table S9. The beta tubulin gene (TUB3) was used as the reference gene for normalizing the expression of the T. pseudonana chitinase genes [71 (link)].