Real time pcr reagents

Manufactured by Thermo Fisher Scientific

Sourced in United States, Australia, Italy

Real-time PCR reagents are a specialized set of laboratory consumables used in the amplification and detection of nucleic acid sequences through the real-time polymerase chain reaction (PCR) technique. These reagents include enzymes, buffers, dyes, and other components necessary for the quantitative analysis of DNA or RNA targets.

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Quantitative real-time PCR reagents Real-time RT-PCR reagents Reagents for real-time PCR Reagents for real time RT-PCR and Western blotting

15 protocols using real time pcr reagents

Steroidogenic Pathway Analysis in Cells

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McCoy's 5A medium and 0.4% trypan blue were purchased from Invitrogen/GIBCO (Carlsbad, CA). Penicillin-streptomycin was purchased from Roche Diagnostics (Indianapolis, IN). BSA and anti-β-tubulin antibody were purchased from Sigma Chemical Co. (St. Louis, MO). Purified hCG was purchased from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). Forskolin was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Rapamycin, anti-mouse or anti-rabbit IgG horseradish peroxidase conjugates, anti-p-S6K1, and anti-S6K1 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-CYP11A1 was obtained from Abcam (Cambridge, MA). Femto Super Signal Chemiluminescence reagent and Restore stripping buffer were purchased from Pierce (Rockford, IL). Progesterone Enzyme Immunoassay (EIA) kit was purchased from Cayman Chemical (Ann Arbor, MI). Real-time PCR reagents, as well as the primers and probes for STAR (assay id: Hs00264912_m1), CYP11A1 (assay id: Hs00167984_m1), HSD3B1 (assay id: Hs01084547_gH), and 18S rRNA (assay id: Hs99999901_s1) were purchased from Applied Biosystems (Foster City, CA). All other reagents used were conventional commercial products.

Moravek M.B., Shang M., Menon B, & Menon K. (2016). HCG-Mediated Activation of mTORC1 Signaling Plays a Crucial Role in Steroidogenesis in Human Granulosa Lutein Cells. Endocrine, 54(1), 217-224.

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Genetic Analysis of Neurotransmitter Pathway SNPs

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SNPs DBH rs1611115, DDC rs6592961, DRD1 rs251937, DRD2 rs4630328, and DRD3 rs167771 were analyzed in this study. Information related to these SNPs is listed in Supplemental Table S1. DNA was extracted from blood cells and analyzed using a TaqMan probe-based PCR approach [24 (link)]. Both TaqMan probes and real-time PCR reagents were purchased from Applied Biosystems (Beijing, China). The catalog numbers are C___2535786_10 for rs1611115, C__29250259_10 for rs6592961, C___3199281_30 for rs251937, C__11339292_10 for rs4630328, and C____949778_10 for rs167771. Real-time PCR was conducted with the ABI7900 real-time PCR instrument following the manufacturer's protocol, described previously [24 (link)]. Briefly, the PCR reactions were performed in a total volume of 15 μl containing 20 ng of genomic DNA, 0.4 μl of the primers and probe mixture, 7.5 μl of universal PCR mix, and water. PCR cycles used were as follows: 95°C for 10 minutes and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results were analyzed using the ABI SDS2.3 software.

Liu Z., Liu J., Zhang Z., Yu H, & Hu F. (2020). Facial Emotion Recognition and Polymorphisms of Dopaminergic Pathway Genes in Children with ASD. Behavioural Neurology, 2020, 6376842.

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Anti-CS-A Monoclonal Antibody Characterization

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Anti-CS-A mAb 2H6 was from Associates of Cape Cod/Seikagaku America (Falmouth, MA, USA). Fluorescence-conjugated, anti-mouse IgM and 5-aza-2′-deoxycytidine (5AzadC) were from Sigma (St. Louis, MO, USA). Primers were from Integrated DNA Technologies (IDT, Coralville, IA, USA). Real-time PCR reagents were from Applied Biosystems (Foster City, CA, USA). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA), FailSafe PCR PreMix Selection kits and Enzyme Mix were from Epicentre Biotechnologies (Madison, WI, USA). Alkaline phosphatase (rAPid) was from Roche (Nutley, NJ, USA).

HERMAN D., LEAKEY T.I., BEHRENS A., YAO-BORENGASSER A., COONEY C.A., JOUSHEGHANY F., PHANAVANH B., SIEGEL E.R., SAFAR A.M., KOROURIAN S., KIEBER-EMMONS T, & MONZAVI-KARBASSI B. (2015). CHST11 gene expression and DNA methylation in breast cancer. International Journal of Oncology, 46(3), 1243-1251.

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Isolation and Osteogenic Potential of Human ASCs

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Human ASC populations (n=15) were isolated from human lipoaspirates under an approved IRB protocol [1 (link), 42 ] and were cultured as described in the supplemental data section. Confirmation of osteogenic potential is also presented in this section (Supplemental Figure 2). Cell culture reagents for maintenance and expansion of ASCs (high glucose DMEM, PBS, antibiotics/antimycotics) were purchased from Mediatech Cellgro (Manassas, VA). Inductive factors for osteogenic differentiation were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). FBS was purchased from Gemini Bioproducts (West Sacramento, CA) or Omega Scientific (Tarzana, CA). Real time PCR reagents were purchased from Applied Biosystems (Foster City, CA). Antibodies to unphosphorylated ERK1/2, JNK1/2 and p38MAPK were purchased from Invitrogen Life Technologies (Carlsbad, CA) and EMD Millipore (Billerica, MA). Polyclonal antibodies to the dual phosphorylated forms of ERK1/2, JNK1/2 and p38MAPK were also purchased from Invitrogen. A monoclonal β-actin antibody was purchased from Abcam (Cambridge, MA). MAPK signaling inhibitors were purchased from EMD Biosciences/Calbiochem (Gibbstown, NJ).

Tsang E.J., Wu B, & Zuk P. (2017). MAPK Signaling has Stage-dependent Osteogenic Effects on Human Adipose-derived Stem Cells in vitro. Connective tissue research, 59(2), 129-146.

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Cell Culture and Real-Time PCR Analysis

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Cell culture medium, serum and supplements, primers and real-time PCR reagents were from Life Technologies (Mulgrave, VIC, Australia). M3G was from Novachem (Collingwood, VIC, Australia). QUANTI-Blue^TM, Normocin^TM, and HEK-Blue^TM Selection reagent were from Jomar Life Research (Scoresby, VIC, Australia). Other reagents were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) unless otherwise specified.

Khabbazi S., Xie N., Pu W., Goumon Y, & Parat M.O. (2016). The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro. Frontiers in Pharmacology, 7, 441.

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Cell Culture and Invasion Assay

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RPMI-1640 medium, Dulbecco’s Modified Eagle Medium, trypsin-EDTA, penicillin/streptomycin, Coomassie brilliant blue R-250, Pierce BCA Protein Assay Kit and real-time PCR reagents were purchased from Life Technologies (Melbourne, VIC, Australia). The 40% acrylamide/bis solution was from Bio-Rad (Gladesville, NSW, Australia). CultreCoat 24 well plates with BME-coated inserts and CultreCoat 96 well medium BME cell invasion assay kits were from Bio Scientific Pty. Ltd (Sydney, NSW, Australia). NeuroCult NS-A proliferation kit (human), heparin solution, human recombinant bFGF and human recombinant EGF were purchased from Stemcell Technologies Australia (Tullamarine, VIC, Australia). Other reagents were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) unless otherwise specified.

Pu W., Qiu J., Riggins G.J, & Parat M.O. (2020). Matrix protease production, epithelial-to-mesenchymal transition marker expression and invasion of glioblastoma cells in response to osmotic or hydrostatic pressure. Scientific Reports, 10, 2634.

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Neuroprotective Pathway Modulation Protocol

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Fludrocortisone, Spiro, DEX, β-amyloid fragment (1–42), wortmannin, rapamycin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), Stemline neural stem cell medium, polyornithine, bovine serum albumin (BSA), and primers for RT-PCR were from Sigma-Aldrich (Milano, Italy). Human b-FGF, penicillin, streptomycin, fungizone, and trypsin were from Life Technologies, Inc. (Invitrogen, Milano, Italy). Quantikine immunoassay caspase-3 Colorimetric kit was provided by Assay Designs, Bologna, Italy. KT5720 was from Biomol Research Laboratory Inc. (DBA, Italy). Rabbit polyclonal antibodies P-Akt (Ser473), P-GSK-3β (Ser9), P-CREB (Ser133), and P-p70S6K (Thr389); mouse monoclonal antibody for P-Tau (Ser396) (PHF13) was from Cell Signalling Technology (Euroclone SpA, Milano, Italy). Total antibodies were from Abcam (Cambridge, UK). RT-PCR and Real-Time PCR reagents were from Life Technologies, Inc. (Invitrogen, Milano, Italy). Primers for RT-PCR were from TibMolBiol (Genova, Italy).

Gesmundo I., Villanova T., Gargantini E., Arvat E., Ghigo E, & Granata R. (2016). The Mineralocorticoid Agonist Fludrocortisone Promotes Survival and Proliferation of Adult Hippocampal Progenitors. Frontiers in Endocrinology, 7, 66.

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Cardioprotective Effects of DHAG on H9c2 Cells

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The cardiomyoblast cell line H9c2 was purchased from Beijing Cell bank (Beijing, China). Fetal bovine serum and RPMI-1640 medium were from Gibco BRL (Gaithersburg, MD, USA). LTA and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caspase-3/9 activity assay kits were from Promega (Madison, WI, USA). Glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), IL-1β, and TNF-α assay kits were purchased from Jiancheng Biological Engineering (Nanjing, China). The cytochrome c assay kit was from R&D systems (Minneapolis, MN, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR, USA). Real-time PCR reagents were purchased from Thermo Fisher (Waltham, MA, USA). DHAG was provided by Prof. Teng from Zhengzhou University.¹⁵ All solvents and chemicals were analytical grade and were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). LTA and DHAG were dissolved in dimethyl sulfoxide (DMSO).

Song Q., Xie X., Hu Z., Xue J., Zhang S, & Xie X. (2020). (Z)-7,4'-dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside attenuates lipoteichoic acid-induced damage in rat cardiomyoblast cells. The Journal of International Medical Research, 48(8), 0300060519889716.

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Mesenchymal Stem Cell Differentiation

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Dulbecco’s modified eagle’s medium (DMEM), Oil red O, alizarin red, dexamethasone, iso butyl methyl xanthine, indomethacin, insulin, β- glycerophosphate, ascorbic acid and basic fibroblast growth factor were purchased from Sigma Aldrich (Steinheim, Germany). Tissue culture plastic plates and flasks were from Eppendorf (Germany). Fluorescent conjugated anti-human antibodies were from BD biosciences (Germany). Fetal bovine serum (FBS) and real-time PCR reagents were purchased from Thermo Fisher scientific (USA).

Somaiah C., Kumar A., Sharma R., Sharma A., Anand T., Bhattacharyya J., Das D., Deka Talukdar S, & Jaganathan B.G. (2018). Mesenchymal stem cells show functional defect and decreased anti-cancer effect after exposure to chemotherapeutic drugs. Journal of Biomedical Science, 25, 5.

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Cardiomyoblast Stress Response Protocol

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H9c2 cardiomyoblast cell line was purchased from Shanghai Cell Bank (Shanghai, China). LTA was purchased from National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Gaithersburg, USA). Caspase-3/9 activity assay kits and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid (MTT) were purchased from Sigma-Aldrich (St. Louis, USA). MDA, GSH, SOD, IL-1β, IL-12, and TNFα assay kits were purchased from Jiancheng Biological Engineering (Nanjing, China). Cytochrome-c immunoassay kit was purchased from R&D systems (Minneapolis, USA). 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and 2’,7’-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (CA, USA). Gamma H2AX (γH2AX) antibody was purchased from BioLegend (San Diego, USA). Real-time PCR reagents were purchased from Thermo Fisher (Waltham, MA, USA). TIM was isolated and identified by Prof. Lin from Shantou University Medical College (Shantou, China) (14 ). All solvents and chemicals used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). LTA and TIM were dissolved in DMSO in the in vitro experiments.

Liu Z., Xie L., Bian T., Qi G, & Wang Z. (2018). (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one reduces lipoteichoic acid-induced damage in rat cardiomyoblast cells. Anatolian Journal of Cardiology, 19(3), 198-204.

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