Minibest universal rna extraction kit

PCC7002 and PCC7002ΔperR cells at log phase with an OD_{730 nm} of 0.6–0.7 were incubated with or without H₂S and H₂S_n (at concentrations of 250 and 500 µM) in sealed centrifuge tubes for 3 h, at 30 °C, and 50 μmol photons·m⁻²·s⁻¹ illumination. H₂S was prepared according to the previous report [44 (link)] and experimental requirements, and the preparation method was as follows: 56.06 mg NaHS was dissolved into 1 mL of buffer (containing 50 mMTris.HCL and 100 μM DTPA), which had been degassed with N₂ prior to NaHS powder solubilization, and diluted according to the desired concentration. Then, the induced cells were harvested by centrifugation at 10,000× g, and 4 °C for 10 min. Total RNA was isolated using the TaKaRa MiniBEST Universal RNA Extraction Kit, and the concentration and quality of RNA were verified by Qubit 4 (Invitrogen, Carlsbad, CA, USA). The cDNA was acquired using the Prime Script™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the SYBR^® Premix Ex Taq™ II kit (TaKaRa, Dalian, China). The primers used for the target genes are shown in Table S1. The reference gene rnpA (SYNPCC7002_A0989) was also included [45 (link)]. The results were analyzed according to the 2^−ΔΔCT method [46 (link)].

Total RNA was extracted from the heart of wild-type and TRIP-Br1^−/− mice by using an RNA extraction kit (Takara Minibest universal RNA extraction kit), and cDNA was synthesized using Superscript II reverse transcriptase from Invitrogen. Quantitative PCR (qPCR) amplifications of various genes were performed using SYBR Green (Takara) and an ABI 7500 fast real-time PCR machine (Applied Biosystems); GAPDH was used as a normalization control. After initial denaturation (30 s, 95°C), amplification was performed using 40 cycles of 5 s at 95°C and 34 s at 60°C. All qPCR data represent means ± SEM. The sequences of the primers used for qPCR are listed below:
AC1: 5′-CCTCGCACTTACTGGTCACA-3′ and 5′-AACCCACGATGTCTGCAAAC-3′;
GAPDH: 5′-CTTGTCATCAACGGGAAGCC-3′ and 5′-CATGAGCCCTTCCACAATGC-3′.

Cells at log phase with an OD₇₃ of 0.6 to 0.7 were harvested by centrifugation at 10,000 × g, 4°C, for 10 min. Total RNA was isolated by using the TaKaRa MiniBEST universal RNA extraction kit, and the concentration of RNA was verified by Qubit 4 (Thermo Fisher). The cDNA was acquired by using the Prime Script RT reagent kit with genomic DNA (gDNA) eraser (TaKaRa, Beijing, China). The SYBR Premix Ex Taq II kit (TaKaRa) was used for qRT-PCR, and the reactions were run in a Light Cycler 480 II sequence detection system (Roche, Shanghai, China). Primers for target genes are all shown in Table S1, and rnpA (SYNPCC7002_A0989) was used as the reference gene (67 (link)). The results were analyzed according to the threshold cycle (2^−ΔΔCT) method (68 (link)).