Power up sybr green kit

RNA was extracted from 0.25×10⁶ cells using Trizol reagent (15596026, Invitrogen). GlycoBlue (5 μg/mL, AM9516, Invitrogen) was added during the isopropanol precipitation step. RNA quality was measured on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). 1 μg RNA was reverse transcribed using high-capacity cDNA synthesis kit (4368813, Applied Biosystems). qPCR used the PowerUP SYBR green kit (A25742, Applied Biosystems), 20 ng cDNA in a 10 μL reaction and primers at 1 μM final concentration (Sequences in Table S5). Primer specificity was ensured by designing primers to span exon-exon junctions, whenever possible, and for each primer pair a melt curve was recorded, compared to in silico predicted melt curves from uMelt (54 (link)) and amplicon sizes analyzed by agarose gel electrophoresis. Recorded Ct values were normalized to the recorded Ct of human HPRT1, and data plotted as ΔCt (Relative expression). To determine absolute expression, defined amounts of linearized plasmid standards were used as PCR template and obtained Ct values used to calculate transcript numbers.

The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used for cDNA generation from 200 ng of RNA with pre-incubation of the RNA template and random hexamers for 10 min at 65°C. Relative quantification of pufM and ppsR transcripts was performed in triplicates in a CFX Connect Real-Time PCR cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the PowerUp Sybr Green kit (Applied Biosystems, USA). The rpoD gene was used as a reference. The comparative C_t method (64 (link)) was used to quantify changes in gene expression. Specific primers and PCR conditions are listed in Table S1.

0.2 mL of Chloroform was added into Eppendorf containing PBMCs and 1 mL of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), then centrifuged at 12,000×g for 15 min at 4 °C to remove debris. The supernatant was collected, placed in a new tube, mixed with isopropyl alcohol, and centrifuged at 12,000×g for 10 min at 4 °C. The supernatant was discarded, and the RNA pellet was washed twice with 1 ml of 75% ethanol. The pellet was air-dried for 30 min, dissolved with 30 μL of RNase-free water, and stored at − 80 °C after spectrophotometric quantification. About 500 ng of RNA was purified and subjected to reverse transcription using the cDNA with High-Capacity Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). Realtime PCR was performed on an Applied Biosystems^® 7500 QuantStudio 5 Real-Time PCR System (Life Technologies; Carlsbad, CA, USA). The qPCR analysis was conducted using a PowerUP SYBR green kit (Thermo Fisher Scientific, Waltham, MA, USA) and the following cycles: 95 °C for 2 min, followed by 95 °C for 15 s and 60 °C for 1 min for 40 cycles. The sequences of primers are reported in Table S3. The relative difference of NQO1, CAT and MT1E gene expression between Ops and CTRs was calculated using 2^-DDCT method and normalized to GAPDH levels as the internal control^{16 (link)}.

Expression studies were performed on samples collected from cabbage, broccoli florets and kale leaves after 1, 6, and 12 days of storage. Relative transcript expression was investigated using three technical replicates from each of the biological replicates from the three time points on day 1, 6 and 12. Genes for ethylene biosynthesis, ACO and ACS, signaling genes EIN2, EIN3 and perception genes like CTR1 and ETR1, and the SAGs, ORE15, NAC29 and SAG12 were analyzed using the primer sequences listed in Table 1. Actin2 was used as an internal reference control (Table 1). Each reaction was 10 µL using SYBR green reagents from Power up SYBR green kit (Thermo-Fisher, Grand Island, NY, USA). Each gene was amplified in three separate reactions using a Thermofisher (Applied biosystems, Foster City, CA USA) thermocycler with the following conditions: 95 °C for 10 min, 45 cycles for 95 °C for 30 s, and 60 °C for 30 s. Relative gene expression was calculated by the ΔCT method. Quantitative PCR experiments were repeated for three times.

HUVECs or exosomes were lysed in Trizol (Invitrogen, USA) and the resulting lysates mixed with chloroform and centrifuged at 13,000 x g for 15 min at 4°C. The aqueous phase (top layer) was collected and mixed with isopropanol to precipitate total RNA. The cDNA was synthesized with 1 μg of total RNA using the SuperScript™ IV VILO™ Master Mix kit according to the manufacturer’s protocol (Thermo Fisher Scientific, Cat. 11756500). Real-time PCR was performed with the PowerUp™ SYBR™ Green kit (Thermo Fisher Scientific, Cat. A25742) according to the manufacturer's instructions. RNA expression was normalized to GAPDH. Relative gene expression was analyzed using the 2ΔΔ-Ct method. All primers are shown in Supplementary Table 1.