Epoch micro volume spectrophotometer system

The saliva-based HBGA binding and blocking assays were performed as described previously [18 (link),26 (link),27 (link)]. To avoid potential HuNoV-specific antibodies in the saliva that may interfere with the receptor-binding assay, saliva samples were boiled before being used in the assays. The boiled human saliva samples with known HBGA phenotypes (A, B, or O) were diluted 1000-fold and coated on 96-well microtiter plates at 4 °C overnight. After blocking with 5% nonfat milk in PBS, HuNoV VLPs were added to a final concentration of 4 μg/mL. The bound VLPs were detected by using serially diluted IgYs (from 1:1000 to 1:128,000), followed by the addition of HRP-conjugated goat anti-chicken IgY at a dilution of 1:5000. The color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (Kirkegaard and Perry Laboratory) and stopped after 10 min of incubation at 22 °C by adding 1 mol/L sulfuric acid. Optical density (OD) was measured at 450 nm with the use of an Epoch Micro-Volume Spectrophotometer System (BioTek, Winooski, VT, USA).

Total RNA was extracted using the TRIzol reagent, as directed by the manufacturer. Quantification of total RNA was conducted with an Epoch microvolume spectrophotometer system (Bio-Tek, Winooski, VT, USA). First-strand complementary (c) DNA synthesis by reverse-transcription (RT) was accomplished using RNase and M-MLV reverse transcriptase to transcribe poly (A)+ RNA with oligo-dT as the primer. Quantitative PCR analysis was carried out using SYBR green. Relative gene expression was quantified using a real-time PCR technique (LightCycler^® 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland). Initial denaturation at 95 °C for 120 s was followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s for all assays. Denaturing at 95 °C for 10 s, cooling to 65 °C for 60 s, and then heated to 97 °C for 1 s in the melting analysis. Each cDNA was amplified using specific primers. The threshold cycle (Ct) values of target genes were normalized to the Ct values of β-actin. We used the comparative Ct method to calculate the fold changes in gene expression. The gene-specific primers used were β-actin as the control group, and the target genes were PPARγ, C/EBPα, SREBP-1c, HSL, ATGL, FAS, and AMPK (Table S1). In the experiment of genes related to lipid metabolism, each group was randomly selected (n = 5) for the independent experiment.