Superscript first strand cdna synthesis system

For tissue expression analysis, leaves, stem tips, hypocotyls, roots, nodules, flower buds, flowers, and pod samples were collected from W82 grown under SD conditions. For rhythmic expression analysis, leaves were collected every 4 h from W82 seedlings under LD or SD conditions at 18 DAE. To compare the expression of FT2a and FT5a in wild‐type and TOE4b overexpression seedlings, leaves were collected at 18 DAE from seedlings grown under LD conditions.
Total RNA was extracted from all the above samples using an Ultrapure RNA kit (Cowin Biotech, Beijing, China). First‐strand cDNA synthesis and removal of genomic DNA contaminants were performed using a SuperScript First‐strand cDNA Synthesis System (Takara, Dalian, China). Quantitative PCR (qPCR) was performed using a Roche LightCycle480 system (Roche, Mannheim, Germany) using a qPCR kit (Roche). β‐Tubulin was used as the internal control. Three independent biological replicates were analysed, and three technical replicate reactions were used for each sample. All qPCR primers are listed in Table S6.

Total RNA was isolated from hippocampal tissue using total RNA extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) adopting the manufacturer’s recommended procedure.
Reverse transcription was performed on an aliquot containing 500 ng of total RNA. The reaction was accomplished on the superscript first-strand cDNA synthesis system, based on the manufacturer’s recommended procedure (Takara, Japan). The sequences of target genes and the specific primers are shown in Table 1.
BDNF, iNOS, and eNOS mRNA levels were measured using a Rotor-Gene 6000 real-time PCR cycler (Corbett, Australia). The Real-time PCR reaction was also performed on 1 μl of synthesized cDNA solution, 5 μl of SYBR Premix Ex Taq II (TliRNase H Plus) (Takara, Japan), and 0.2 μl of each primer. Forty amplification cycles consisted of denaturation at 95°C for 15 s, annealing according to Table 1 for 30 s and an extension at 72°C for 30 s. Each sample was analyzed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as a loading control to normalize target gene expression. Relative expression levels of the target cDNAs were quantified by the 2ΔΔCts method.