Primerscript reverse transcriptase

To analyze the expression pattern of OsHMGB707, seedlings of the rice cultivar Nipponbare (Oryza sativa L. ssp japonica) at the four-leaf stage were subjected to various treatments, including dehydration (water withholding), salt (150 mM NaCl), oxidative stress (1% H₂O₂), cold (4°C), and heat (42°C). Also, plant hormones like 0.1 mM abscisic acid (ABA), and jasmonic acid (JA) were separately sprayed on the seedlings. The roots were also submerged into the solution and then sampled at designated times.
Total RNA was extracted using TRNzol reagent (TIANGEN, DP424, China), and cDNA synthesized using PrimerScript reverse transcriptase (TaKaRa, RR036A, Japan). Quantitative PCR (qPCR) was performed in a 96-well plate with a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, United States) using the SYBR premix Ex Taq (TaKaRa, RR820A, Japan) according to the manufacturer’s instructions. The reaction conditions were as follows: 95°C for 60 s, followed by 40 cycles at 94°C for 15 s and 62°C for 60 s. The rice actin gene OsACT2 (Os11g0163100) was used as the reference gene to normalize the target gene expression, calculated using the relative quantification method (2^–ΔΔCT).

Total RNA was extracted with Trlzol® reagent (Thermo Fisher Scientific, Inc.) and reversely transcribed into complementary DNA (cDNA) by PrimerScript reverse transcriptase (Takara, Tokyo, Japan). Subsequently, SYBR Premix Ex Taq reagent (Takara, Tokyo, Japan) was applied to perform qPCR reaction on an ABI 7500 quantitative PCR instrument (ABI/Perkin Elmer, CA, USA). At last, the relative gene expression was determined with the adoption of 2^−ΔΔCt methods [22 (link)].

Total RNAs (1-2 μg) were reverse-transcribed at 42°C using PrimerScript™ Reverse Transcriptase (Takara Bio, Shiga, Japan). Then iTaq™ Universal SYBR^® Green Supermix (Bio-rad, Hercules, CA, USA) was employed to perform quantitative PCR on a CFX Connect™ Real-Time PCR Detection System (Bio-rad). Gene expressions were determined using the 2^−ΔΔCt method, normalizing to housekeeping genes GAPDH or ACTB. Oligo sequences for qRT-PCR: GAPDH (forward: 5′-AAT CAA GTG GGG CGA TGC TG-3′, reverse: 5′-TGG TTC ACA CCC ATG ACG AA-3′); ACTIN (forward: 5′-CTC TTC CAG CCT TCC TTC CT-3′, reverse: 5′-AGC ACT GTG TTG GCG TAC AG-3′); HMGA1 (forward: 5′-ACT GGA GTC TCC TGT GGT GTG T-3′, reverse: 5′-AGT GCT ATT TCC CCT CCC TTC-3′); HMGA2 (forward: 5′-CAC TTC AGC CCA GGG ACA AC-3′, reverse: 5′-GCC TCT TGG CCG TTT TTC TC-3′); ACTA2 (forward: 5′-CAA TGA GCT TCG TGT TGC CC-3′, reverse: 5′-GCA AGG CAT AGC CCT CAT AGA-3′); SOX2 (forward: 5′-CAC AAC TCG GAG ATC AGC AA-3′, reverse: 5′-CGG GGC CGG TAT TTA TAA TC-3′); FOXM1 (forward: 5′-AGT AGT GGG CCC AAC AAA TTC AT-3′, reverse: 5′-CTT TTG GCA TCA TAG CTG GTT TG-3′); CYR61 (forward: 5′-GGT CAA AGT TAC CGG GCA GT-3′, reverse: 5′-GGA GGC ATC GAA TCC CAG C-3′); PLAU (forward: 5′-TGT GAA GCT GAT TTC CCA CCG-3′, reverse: 5′-GCC TTG GAG GGA ACA GAC GAG-3′).

Total RNA was isolated using Trizol reagent (Invitrogen, 15596018), according to the manufacturer’s instructions. In all, 1 μg RNA was primed with random hexamers and reverse transcribed with PrimerScript Reverse Transcriptase (Takara, Kusatsu, Japan, 6110). The following primers were used: NDRG1, forward: 5′-AAGATGGCGGACTGTGGC-3′ reverse: 5′-TCAGGCGGGTCATGCTAG-3′. GAPDH, forward: 5′-TGAACGGGAAGCTCA-3′ reverse: 5′-TCCACCACCCTGTTGCTGTA-3′. These primers were synthesized by Sangon Biotech Co., Ltd. All samples were estimated by normalization to GAPDH expression levels.