Genomelab gexp start kit

EBN was purchased from Blossom View Sdn. Bhd (Terrengganu, Malaysia) while simvastatin was purchased from Hangzhou MSD Pharmaceutical Co., Ltd (Hangzhou, China). Standard rat pellets were purchased from Specialty Feeds (Glen Forrest, WA, USA), cholesterol was purchased from Amresco (Solon, OH, USA), cholic acid was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and palm oil was purchased from Yee Lee Edible Oils Sdn. Bhd. (Perak, Malaysia). Analytical grade ethanol was purchased from Merck (Darmstadt, Germany), while RCL2 solution was purchased from ALPHELYS (Toulouse, France). Lipid profile kits were purchased from Randox Laboratories Ltd. (Crumlin, County Antrim, UK), while an oxLDL ELISA kit was purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China). An RNA extraction kit was purchased from RBC Bioscience Corp. (Taipei, Taiwan), and a GenomeLab™ GeXP Start Kit was purchased from Beckman Coulter Inc (Miami, FL, USA).

Brown rice (BR) of Malaysian mixed varieties (MR219 and MR220) was procured from Padiberas Nasional (BERNAS) factory, Seri Tiram Jaya, Selangor. Food grade H₂O₂ and sodium hypochlorite (NaOCl) were purchased from Bendosen Laboratory Chemicals (Selangor, Malaysia) and from Dexchem Industries Sdn. Bhd. (Penang, Malaysia), respectively. Human SH-SY5Y neuroblastoma cell line was purchased from the American Type Culture Collection (ATCC), USA. Dulbecco's Modified Eagle's Medium/Nutrient F-12 mixture, fetal bovine serum, gentamicin, phosphate-buffered saline (PBS), human Aβ(1-42), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Analytical grade H₂O₂ was purchased from Merck (Darmstadt, Germany), while CytoTox96 Non-Radioactive Cytotoxicity Assay was purchased from Promega (Southampton, UK). The GenomeLab GeXP Start Kit was purchased from Beckman Coulter, Inc. (Miami, FL, USA), and the Total RNA Isolation Kit was purchased from RBC Bioscience Corp. (Taipei, Taiwan). Magnesium chloride (MgCl₂), DNA Taq polymerase, HPLC grade water, acetic acid, acetonitrile, methanol, and isopropanol were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA).

Reverse transcription reaction mixture was prepared according to the GenomeLab GeXP Start Kit (Beckman Coulter, USA). Briefly, 11 μL of RNA-free water was mixed with 2 μL of customized reverse primer of the desired gene, 1 μL of reverse transcriptase, 4 μL of reverse transcription buffer and 1 μL of 50 ng/μL of RNA sample isolated previously. The reverse transcription reaction was performed as follow: primer annealing at 48°C for 1 minute, reverse transcription at 42°C for 60 minutes and denaturation at 95°C for 5 minutes. Subsequently, the cDNA product was amplified by polymerase chain reaction (PCR) reaction. The PCR reaction mixture, which consists of 4 μL of 5X PCR buffer, 4 μL of MgCl₂, 2 μL of customized forward primer mixture (Table 1), 0.7 μL of Taq polymerase and 9.3 μL of cDNA was prepared. Next, PCR was performed as follow: denaturation of DNA at 95°C for 10 minutes, DNA annealing at 94°C and 55°C, both for 30 seconds, and elongation of DNA at 70°C for 1 minute. The second and third step were repeated for a total of 35 thermal cycles. Lastly, the PCR tube with sample was hold at 4°C.