Revertra ace reverse transcriptase kit

Total RNA from rice prepared for the microarray analyses as described by Aung et al. (2018) (link) was used to confirm the expression patterns of the genes OsDMAS1 and OsNAAT1 under Fe excess conditions by quantitative real-time polymerase chain reaction (RT-PCR) analyses. For the expression analyses of OsNAS3 knockout and NT plants from ×1 Fe and ×70 Fe cultures, rice RNA was extracted from hydroponically grown leaves. For all samples, the first-strand cDNA was synthesized using the ReverTra Ace reverse transcriptase kit (Toyobo, Osaka, Japan) and oligo-d(T)₃₀ primers. Then, qPCR was performed in a StepOnePlus^TM Real-Time PCR System (Life Technology, Tokyo, Japan) with SYBR Premix Ex Taq II reagent (Takara, Shiga, Japan). The transcript abundance was normalized against the rice alpha-tubulin transcript level. The primer sequences used for gene expression analyses were as follows: 5′GCC GGC ATC CCG GCA GCG GAA GAT CA 3′ for OsDMAS1 FW and 5′ CTC TCT CTC TCG GGC ACG TGC TAG CGT 3′ for OsDMAS1 RV; 5′-TAAGAGGATAATTGATTTGCTTAC-3′ for OsNAAT1 FW and 5′-CTGATCATTCCAATCCTAGTACAAT-3′ for OsNAAT1 RV; 5′ CGA TGA CTG CTT CCA TCG CTT G 3′ for OsNAS3 FW and 5′ GGC A TG CAT TCA TGC ATG ACT GC 3′ for OsNAS3 RV; 5′ TCT TCC ACC CTG AGC AGC TC 3′ for alpha-tubulin FW and 5′ AAC CTT GGA GAC CAG TGC AG 3′ for alpha-tubulin RV.

Total RNA from zebrafish tissues and embryos was extracted with TRIzol (Invitrogen, USA) according to the manufacturer's instructions. The obtained RNA was analyzed by agarose gel electrophoresis, and its concentration was measured using a spectrophotometer (Thermo, USA). Then, 1 µg of RNA was removed for DNA removal, and a kit (Toyobo, Japan) was used for reverse transcription. cDNA was obtained using the ReverTra Ace reverse transcriptase kit (Toyobo, Japan) and random primers. The fluorescence quantitative PCR assay was performed using the SYBR green mix (Toyobo, Japan) according to the manufacturer's instructions: 2×SYBR Green real-time master mix, 10 µL; cDNA, 2 µL; F, 0.4 µL; R, 0.4 µL; H₂O, 7.2 µL. The PCR amplification conditions were: 95 °C for 3 min; 45 cycles × (95 °C for 15 s; 55 °C for 20 s; 72 °C for 30 s); 65 °C for 0.06 s; 95 ℃, 0.5 s. The primers were listed in Figure S1.

To validate the accuracy of transcriptome data, the expression of DEGs was verified by real-time PCR. Total RNA was extracted, as per manufacturer instructions. After genomic DNA removal by DNase I, cDNA was synthesized using the ReverTra Ace reverse transcriptase kit (Toyobo, FSK-100, Osaka, Janpan) and random primers. Gene expression was quantified by real-time PCR using the SYBR Green Mix (Toyobo, QPK-201), according to manufacturer instructions; the reaction mixture comprised 10 µL 2 × SYBR Green Real-Time Master Mix, 2 µL cDNA, 0.4 µL forward and reverse primers each, and 7.2 µL H₂O. The cycling conditions were as follows: 95 °C for 3 min; 45 cycles × (95 °C for 15 s; 55 °C for 20 s; 72 °C for 30 s); 65 °C for 0.06 s; and 95 °C for 0.5 s. Primers were designed by Primer5 and sequences are listed in Table S4.