RT-PCR reagents were purchased from Invitrogen and primers (supplied by Sigma Aldrich) are described by Van Dongen et al. [22 (link)] and in electronic supplementary material, table S1. Reverse transcription (RT) was performed using 500 ng of total PBMC RNA mixed with 1 μl JH reverse primer (10 μM), 1 μl dNTPs (0.25 mM) and RNase-free water added to make a total volume of 11 μl. This was incubated for 5 min at 65°C, and 4 μl First strand buffer, 1 μl DTT (0.1 M), 1 μl RNaseOUT™ Recombinant Ribonuclease Inhibitor and 1 μl SuperScript™ III reverse transcriptase (200 units μl−1) was added. RT was performed at 50°C for 60 min before heat-inactivation at 70°C for 15 min. PCR amplification of cDNA (5 μl of the RT product) was performed with the JH reverse primer and the FR1 forward primer set pool (0.25 μM each), using 0.5 μl Phusion® High-Fidelity DNA Polymerase (Finnzymes), 1 μl dNTPs (0.25 mM), 1 μl DTT (0.25 mM), per 50 μl reaction. The following PCR programme was used: 3 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C, with a final extension cycle of 7 min at 72°C on an MJ Thermocycler.
Rt pcr reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
RT-PCR (Reverse Transcription Polymerase Chain Reaction) reagents are a collection of chemical compounds and enzymes used to detect and amplify specific RNA sequences. These reagents enable the conversion of RNA into complementary DNA (cDNA) and the subsequent exponential amplification of the target genetic material through a series of temperature-controlled cycles. The core function of RT-PCR reagents is to facilitate the sensitive and accurate detection and quantification of RNA molecules, making them a critical tool in various applications, such as gene expression analysis, disease diagnosis, and viral detection.
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Lab products found in correlation
Prism 7500, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Culture media, by Thermo Fisher Scientific (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Dsp virus pathogen mini kit, by Qiagen (1 mentions)
Absolute qpcr sybr green rox mix, by Thermo Fisher Scientific (1 mentions)
7900 ht real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Basic fgf, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Rna extraction kit, by Qiagen (1 mentions)
13 protocols using rt pcr reagent
1
RT-PCR for Immune Cell Profiling
2
Biochemical Reagents for Cell Culture
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Enhanced chemiluminescence reagents were obtained from Amersham Biosciences (Piscataway, NJ, USA); SDS-PAGE supplies from Bio-Rad (Richmond, CA, USA); ATP, common reagents, and salts from Sigma (St. Louis, MO, USA); culture media, penicillin, streptomycin, Hanks’ balanced salt solution, L-glutamine, agarose, and RT-PCR reagents from Invitrogen (Carlsbad, CA, USA); fetal bovine serum from Hyclone (Logan, UT); Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN Biomedical (Aurora, OH, USA); forskolin from Calbiochem (La Jolla, CA, USA). Krebs–Ringer bicarbonate (KRB) buffer contained (in mM) 25 HEPES (pH 7.4), 115 NaCl, 24 NaHCO3, 5 KCl, 1 MgCl2, and 2.5 CaCl2.
3
Ly6G Antibody Immunofluorescence Protocol
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Rat monoclonal Ly6G antibody (RB6-8C5) was purchased from Abcam (Cambridge, MA, USA). Fluorescein isothiocyanate- (FITC-) labeled goat anti-rat IgG conjugate and RT-PCR reagents were purchased from Invitrogen (Carlsbad, CA, USA). Other biochemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. The oligonucleotide primers for LTB4DH and β-actin were obtained from Genome Research Centre, The University of Hong Kong.
4
Quantifying HIV-DNA in PBMCs
5
Quantification of Total HIV DNA
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Total HIV DNA was quantified from PBMC at the enrollment time point for all patients using an in-house, real-time quantitative PCR assay as previously described [31 (link)]. Briefly, total nucleic acid was extracted from PBMCs on the Qiasymphony platform using the DSP virus/pathogen mini kit (Qiagen) according to the manufacturer’s protocol. An ABI PRISM 7500 real-time PCR instrument with Invitrogen RT-PCR reagents was used for amplification and detection of HIV-1 DNA. The HIV DNA copies were calculated as HIV-1 copies per 10^6 haploid pyruvate dehydrogenase (PDH) copies. Results are reported as copies of HIV per million cells [59 (link)].
6
Culturing Human Cerebral Endothelial Cells
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The human cerebral microvascular endothelial cells/clone D3 (hCMEC/D3) were grown in EBM-2 medium (Lonza, Switzerland) supplemented with ascorbic acid, hydrocortisone, basic FGF (Sigma-Aldrich, France), foetal bovine serum (PAA, France) and hepes (Invitrogen, France). Cells were grown on 6-well plates (Corning, France) coated with rat tail collagen type-I (BD Biosciences, France). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) was obtained from LGC Promochem (Molsheim, France). Control-siRNA (Neg.
siRNA AF 488, reference 1027284), SiRNA for AhR (Hs_AHR_5, reference SI02780148), HiPerFect transfection reagent and RNA extraction kits were purchased from Qiagen (Qiagen, Germany). RT-PCR reagents were purchased from Invitrogen (Invitrogen, France). Primers were synthesized by Invitrogen Life Technologies (Invitrogen, France). The Power SYBR Green PCR Master Mix was from Applied Biosystems (Applied Biosystems, France) and the ABsolute QPCR SYBR Green ROX Mix from Abgene (Thermofisher scientific, France).Equipment used: a nucleic acid spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, USA), a programmable thermal cycler (PTC-100 programmable thermal controller, MJ research Inc., USA) and a 7900 HT Real-Time PCR Detection System (Applied Biosystems, Foster City, CA).
siRNA AF 488, reference 1027284), SiRNA for AhR (Hs_AHR_5, reference SI02780148), HiPerFect transfection reagent and RNA extraction kits were purchased from Qiagen (Qiagen, Germany). RT-PCR reagents were purchased from Invitrogen (Invitrogen, France). Primers were synthesized by Invitrogen Life Technologies (Invitrogen, France). The Power SYBR Green PCR Master Mix was from Applied Biosystems (Applied Biosystems, France) and the ABsolute QPCR SYBR Green ROX Mix from Abgene (Thermofisher scientific, France).Equipment used: a nucleic acid spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, USA), a programmable thermal cycler (PTC-100 programmable thermal controller, MJ research Inc., USA) and a 7900 HT Real-Time PCR Detection System (Applied Biosystems, Foster City, CA).
7
Bioactive Bone Substitute Characterization
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Cerament (HA-CS) and Cerament G (HA-CS-G) were supplied by Bone Support AB, Lund, Sweden. 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT), Sigmafast p-Nitrophenyl phosphate (pNPP), Dulbecco's Modified Eagle's Medium - high glucose (DMEM - HG), foetal bovine serum (FBS), antibiotic cocktail (penicillin-streptomycin), TRIzol Reagent and primers for real-time polymerase chain reaction (RT-PCR) were purchased from Sigma Aldrich (St. Louis, Missouri). Mouse monoclonal COLI, monoclonal OPN and rabbit polyclonal RunX2, polyclonal OCN, goat anti-rabbit Alexa Fluor 488 (AF488) and goat anti-mouse Cy3 secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Paso Robles, California) and Sigma Aldrich. Mouse monoclonal COLI, OCN and OPN and polyclonal bone sialoprotein (BSP) antibodies, DRAQ5, goat anti-mouse Alexa Fluor 488 (AF488) were procured from Abcam (Cambridge, United Kingdom). RT-PCR reagents were purchased from Thermo Scientific (Waltham, Massachusetts). Rat BMP-2 and BMP-7 Enzyme-linked immunoabsorbance assay (ELISA) kits were purchased from Abnova Inc. (Taipei City, Taiwan and Qayee-Bio, Shanghai, China) respectively. All basic reagents (such as sodium chloride) were of high purity and purchased from recognised suppliers.
8
Metformin, DMBA, and TCDD Effects
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Metformin, DMBA and TCDD were obtained from Toronto Research Chemicals (Toronto, ON, Canada). TRIzol, cDNA Reverse Transcription kit, SYBR Green, and all RT-PCR reagents were obtained from ThermoScientific (Foster City, CA, USA). Western blot reagents, detection kits, and acrylamide/bisacrylamide were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Primary and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada).
9
Ghrelin, Neurotransmitter Assay Protocol
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All solutions were prepared on the day of the experiment. Ghrelin was purchased from Phoenix Pharmaceuticals (Belmont, CA, United States); all RT-PCR reagents were purchased from Applied Biosystems (Foster City, CA, United States); bicuculline methiodide, kynurenic acid and all salts used for the preparation of aCSF, slicing solution and intracellular solution were purchased from Sigma Pharmaceuticals (Oakville, ON, Canada).
10
Glucose Metabolism in Cell Culture
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High glucose Dulbecco’s modified eagle medium and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, United States), Trizol was from Life Technologies, primer probes and real-time polymerase chain reaction (RT-PCR) reagents were from Applied Biosystems by Thermo Fisher Scientific, CA , United States. Other reagents, all analytical grade quality, were from Sigma (St. Louis, MO, United States).
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