T100 thermal cycler system

To extract total RNA from sp-classic, sp-2798, and sp-5732, shoot apices were collected between 13 and 17 days after germination (DAG) from plants grown in a greenhouse. As defined in a previous report [24 (link)], transitional meristems (TM) and sympodial shoot meristems (SYM) were imaged using a stereoscope. More than 30 meristems were dissected and collected from shoot apices fixed by acetone fixation for RNA stabilization, as previously reported [24 (link)]. Total RNA was extracted using the PicoPure RNA Extraction kit (Arcturus) and treated with the RNase-Free DNase Set (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. One microgram of total RNA was used for cDNA synthesis using ReverTra Ace-α^® (TOYOBO, Osaka, Japan). RT-PCR was performed using i-Taq^TM DNA Polymerase (Intron) and a T100^TM Thermal Cycler system (Bio-Rad, Hercules, CA, USA). Real-time quantitative RT-PCR (qRT-PCR) was used to verify the expression of biomarkers in the sp alleles. Two biological replicates of TM and SYM were used for qRT-PCR, and the expression values were analyzed using the CFX96TM Real-time PCR System (Bio-Rad, Hercules, CA, USA). The threshold cycle (Ct) values were calculated and normalized against Ubiquitin using the StepOne™ software v2.3 (Applied Biosystems, Ltd., Waltham, MA, USA). Primer information is shown in Table S8.

The total RNA of cells was extracted using an RNA extraction kit (Promega, LS1040, USA) according to the manufacturer’s instruction. The RNA concentration was measured with a NanoDrop One instrument (Thermo Fisher Scientific, USA). The cDNA was prepared using the Eastep™ RT Master Mix (5X) (Promega, LS2050, USA). qRT-PCR was performed using GoTaq^® qPCR Master Mix (Promega, A6001, USA) in T100TM Thermal Cycler System (BioRad, USA). Relative gene expression was normalized by GAPDH for osteogenesis and apoptosis using a 2^−ΔΔCt method. The primers are listed in Supplemental data 2.

Leptin Receptor^db mice lack the functional, full-length Ob-Rb leptin receptor. Two microliters of HotSHOT DNA was combined with 23 mL of the PCR mixture. A total of 500 nM of each primer (forward: 5′-AGAACGGAC ACTCTTTGAAGTCTC-3′; reverse: 5′-CATTCAAACCATA GTTTAGGTTTGTGT-3′) was combined with PCR buffer, 2 mM MgCl₂, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, and 0.2 mM dTTP (KAPA2G Robust HS; Kapa Biosystems, Cape Town, South Africa) to obtain a volume of 25 μL. Amplification was performed with a T100^TM Thermal Cycler system (Bio-Rad, Singapore). The PCR product obtained with the 25-μL mixture was digested by the direct addition of 25 μL of 1 × digestion cocktail containing 19 μL of water, 5 μL of 10 × CutSmart Buffer (New England Biolabs, Ipswich, MA, United States), and 1 μL of RsaI restriction enzyme (New England Biolabs) and overnight incubation of the mixture at 37°C. The digested products (50 μL) were analyzed in 4% agarose (Takara Biomedicals, Osaka, Japan) with 1 × TAE buffer containing 0.05% (v/v) GoldView^TM. Digestion with RsaI yielded 135-bp fragments in + / + mice and 135-, 108- and 27-bp fragments in heterozygotic db/ + mice.

Total RNA was isolated from the frozen tissues using Takara RNAiso Plus (Takara Bio. Inc., Otsu, Shiga, Japan) according to the manufacturer's protocol. Genomic DNA (gDNA) was removed and cDNA was reverse-transcribed using Takara PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara Bio. Inc., Otsu, Shiga, Japan) in a T100^TM Thermal Cycler System (BioRad, USA). The experimental protocol utilized was first gDNA removal (42°C, 2 min), followed by reverse transcription (37°C 15 min, 85°C 5 s). Subsequently, all samples were amplified by a 25 μl reaction volume in a CFX96^TM Real-time PCR Detection System (BioRad, USA), using SYBR^® Premix Ex Taq^TM II (Takara Bio. Inc., Otsu, Shiga, Japan). All samples were run in triplicate. The seven identified genes were investigated. The amplification program was repeated for 40 cycles. Primer sequences are shown in Table 1. For relative quantification, gene expression was normalized to expression of GAPDH housekeeping gene and compared by 2^−ΔΔCT method.

To extract total RNA from MKN-45 cells, we applied the Trizol reagent to separate the whole RNA according to the manufacturer’s directions (GeneAll Biotechnology, Korea). Subsequently, the extracted RNA (1 μg) was used to synthesize cDNA using its particular kit (Biofact, Daejeon, South Korea) via BioRad T100 thermal cycler system. Then, to evaluate B7-H7, Caspase3-8-9, BCL-2, and BAX genes expression, StepOne Plus qRT-PCR Device (Applied Biosystems, Foster City, USA) was utilized to carry out real-time PCR. GAPDH was served as an internal control to assess expression of genes. Also, all reactions were triplicated. For qRT-PCR results, 2^-∆∆Ct method was utilized to compare the downregulation or upregulation of targeted genes in transfected or treated cells with control cells (control is assumed as 1). Gene-specific primers sets are presented in Table 2.

RT‒PCR analysis was performed as previously described^{36 (link)}. Total RNA was extracted from adult fly heads using an Easy-Blue system (iNtRON Biotechnology). cDNAs were synthesized from 3 µg of total RNA using GoScript Reverse Transcription (A2791; Promega) following the manufacturer’s standard protocol. For RT‒PCR analysis, each target gene was amplified with the corresponding primer set (Supplementary Table 1) using GoTaq G2 Master Mix (M7823; Promega) in a C1000 Thermal Cycler, C1000 Touch Thermal Cycler, or T100 Thermal Cycler system (Bio-Rad).