Thermocycler

The thermal reactions were performed in 25 µL reaction volumes containing about 25–50 ng of template DNA, 2 µL of 10X buffer containing 15 mM MgCl2, 0.2 µM each of forward and reverse primer, 2 µL of 2 mM dNTPs, and 0.2 µL of 1 U of Taq DNA polymerase (Invitrogen USA). The amplifications were performed in a Thermo cycler (MJ Research, USA) programmed for an initial denaturation of 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s of 45°C annealing temperature, and extension of 1.0 min at 72°C, with a final extension of 10 min at 72°C and a final hold at 4°C. The amplicons of PCRs were resolved on 2.5% super fine resolution agarose gels (SFR, Amresco), along with the standard size 100 bp marker at 100 V, and documented in the Bio-Rad gel documentation unit.

The genomic SSRs used for genotyping and bulk segregant analysis were obtained from the earlier reports [8 (link)]. The bulk segregant analysis was performed with 300 genomic and 8 genic SSR markers on bulk DNA of dura and pisifera genotypes. The thermal reactions were performed in 25 μL reaction volume containing about 25–40 ng of template DNA, 2 μL of 10X buffer having 15 mM MgCl2, 0.2 μM of each forward and reverse primer, 2 μL of 2 mM dNTPs, 0.2 μL of 1 U of Taq DNA polymerase (Invitrogen, USA). The amplifications were performed in a Thermo cycler (MJ Research, USA) programmed for an initial denaturation of 3 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s of 45°C annealing temperature, extension of 1.0 min at 72°C, with a final extension of 10 min at 72°C, and final hold at 4°C.

The endothelial cell biosensor assay (Figure 1) was conducted as previously described [13 (link),16 (link)]. Briefly, hCAECs (Lonza, Allendale, NJ) were seeded in a 24-well plate and grown to confluence in complete media (Lonza). The cells were then serum-starved with Basal Media (Lonza) for 24 hours prior to exposure. Confluent hCAEC were incubated with 10% serum obtained from coronary artery disease (CAD) patients (ie., 50 μl serum in 450 μl media, per well) or 10% serum obtained from otherwise healthy individuals. Each plate of cells was treated with approximately equal numbers of subjects from each group in a randomized, blinded fashion. The samples were incubated for 4 hours at 37°C. RNA was extracted from the cells and cDNA was made using a ThermoCycler (Model # PTC-200; MJ Research). The cDNA was used to determine gene expression via quantitative polymerase chain reaction (LightCycler 480 II, Roche, Indianapolis, IN). Specific targets included interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), with TATA-box protein used as a housekeeping gene.Figure 1

Endothelial cell biosensor approach. Primary coronary artery endothelial cells are incubated with 10% serum from individual patients for 4 h, followed by isolation of RNA, and then assayed for transcriptional responses.

LAMP primers were found from literature^{15 (link)} and their oligonucleotide sequences can be found in Supplementary Table S1. They were purchased from Sigma-Aldrich (St. Louis, MO, USA). 10× primer sets were created to contain 16 µM each of FIP and BIP primers, 8 µM each of Loop-F and Loop-B primers, and 2 µM of F3 and B3 primers. LAMP reactions were prepared on ice and utilized the WARMSTART LAMP Kit DNA & RNA (E1700; New England Biolabs Inc, Ipswich, MA, USA). The final LAMP mixture contained 5:1:0.4:1:2.6 ratio of Warm Start LAMP 2× master mix, 10× primer mix, target bacteria dilution (or nuclease-free water for no target control, NTC), 20 mg/mL bovine serum albumin (B8667; Sigma), and nuclease-free water. Conventionally amplified samples were conducted in a thermocycler (MJ Research, Waltham, MA, USA) programmed to run at 65 °C for 30 min, followed by a refrigeration step occurring at 4 °C.

PCR was performed on the resultant cDNA from each sample using adiponectin, adipoR1, adipoR2, and β-actin primers. All of the primers were prepared by Mission Biotech (Taipei, Taiwan). The amplification was performed using a thermocycler (MJ Research, Waltham, MA, USA). The 25 μL reaction mixture consisted of 5 μL of cDNA, 1 μL of sense and antisense primers, 200 μM of each deoxynucleotide (DTT), 5 μL of 10x Taq polymerase buffer, and 1.25 U of GoTaq polymerase (Promega, Midison, WI, USA). The PCR reaction was performed using an annealing temperature of 56°C with GoTaq polymerase, cDNA, and the following primers: sense 5′-AATCCTGCCCAGTCATGAAG and antisense 5′-GGAACATTGGGGACAGTGC for adiponectin; sense 5′-AGACCACCTATGCCCTCCTT and antisense 5′-GCTGTGGGGAGCAGTAGAAG for adipoR1; sense 5′-TGGGAAGTTTTGTTCCTTGG and antisense 5′-TAGAGGGCAGCTCCTGTGAT for adipoR2; and sense 5′-CTGGAGAAGAGCTATGAGCTG and antisense 5′-AATCTCCTTCTGCATCCTGTC for β-actin. The DNA fragments were amplified for 25–30 cycles (30 sec at 94°C; 1 min at 50–52°C; 1 min at 72°C) followed by a final 7 min extension step at 72°C. The products were subjected to electrophoresis on a 1.5% agarose gel and analyzed using a gel analyzer system. Each mRNA level was normalized relative to the β-actin mRNA levels.

All experiments for LAMP assay were run in triplicate. The LAMP reactions were performed using WarmStart^TM Colorimetric LAMP 2X Master Mix (New England Biolabs, Hitchin, UK). A 10X primer mix (FIP, 16 µM; BIP, 16 µM; F3, 2 µM; B3, 2 µM; LF, 4 µM; LB, 4 µM) was prepared. A 25 µL reaction mixture (12.5 µL 2X MasterMix; 2.5 µL 10X primer mix; 2.5 µL RNA and 7.5 µL DNase & RNase-free molecular grade water) was mixed homogeneously and centrifuged. The LAMP assays were performed in a thermocycler (MJResearch) at 65 °C for 30 min or in the engineered device. Colour change was observed directly by the naked eye or through AI image processing and agarose gel electrophoresis was performed to confirm the results. The completion of amplification was indicated by the colour in the tube, wherein yellow was considered positive and pink was regarded as negative. All amplicons were confirmed by 2% agarose gel electrophoresis.