Dntp mix

Manufactured by Meridian Bioscience

Sourced in United Kingdom, United States

The DNTP mix is a laboratory reagent that contains a mixture of the four deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) necessary for DNA synthesis and amplification. This product provides the building blocks required for various molecular biology applications, such as PCR, DNA sequencing, and DNA labeling.

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15 protocols using dntp mix

RNA Extraction and RT-PCR Analysis

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RNA was extracted from E14.5 freshly dissociated and purified ENCCs, and from adult whole brain as described above. The concentration of total RNA in each sample was measured using a NanoDrop ND-1000 spectrophotometer. cDNA was synthesised using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad); 100-350ng of total RNA was used in a final reaction volume of 20 μl according to the manufacturer’s instructions. Control reactions using no reverse transcriptase or substituting cDNA with water were run in parallel for each tissue.
RT-PCR was conducted using intron-spanning specific primer pairs (S1 Table) and a touchdown PCR (TD-PCR) cycling program (S2 Table). A standard RT-PCR protocol was used consisting of MangoTaq (0.2 μl, Bioline), dNTP Mix (0.8 mM, Bioline), MgCl₂ (2 mM, Bioline), and primer pairs (1 μM each) in a final reaction volume of 20 μl. RT-PCR products (5–15 μl) were resolved by gel electrophoresis on a 1–2% agarose gel, containing either ethidium bromide (0.06 μg, Sigma) or GelRed (0.5x, Biotium), together with 100 bp DNA Ladder (Life Technologies) or Ready-to-Use 100 bp DNA Ladder (Biotium) to estimate product size. Control reactions were run in parallel for each RT-PCR reaction.

Hirst C.S., Foong J.P., Stamp L.A., Fegan E., Dent S., Cooper E.C., Lomax A.E., Anderson C.R., Bornstein J.C., Young H.M, & McKeown S.J. (2015). Ion Channel Expression in the Developing Enteric Nervous System. PLoS ONE, 10(3), e0123436.

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PBMC Sorting for hILC RNA-seq Analysis

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PBMCs from 3 DF and 5 DHF patients at febrile and convalescent phases were sorted for hILCs using a FACS Aria III instrument (BD Biosciences) for hILCs RNA-seq and qPCR experiments. PBMCs were stained with Lineage cocktail antibodies (CD3, CD14, CD16, CD19, CD20, CD56) (FITC) and CD127 (APC/Cy7). After staining, cells were washed and resuspended in a cold FACS buffer (2% FBS and 0.25 mM EDTA in PBS). Immediately before sorting, cells were filtered through a 35-μm strainer. Helper ILCs were gated as Lin^-CD127⁺ cells within the lymphocyte gate (FSC^lowSSC^low) after exclusion of doublets and sorted directly into in 96-well plates with 4 μl of lysis buffer composed of 0.4% Triton-X 100 (Calbiochem, 648466), recombinant RNase inhibitor (Clonetech, 2313B), and 10 mM dNTP mix (Bioline, 39053). Sorting mode was set to single cell resolution in order to gain the highest purity. Fifty hILCs were sorted into each well. Approximately 3-5 wells were obtained per sample. Plates were immediately spun down after sorting, then sealed and snapped frozen with dry ice before moving to -80°C freezer for storage until further processing within 2-3 days.

Poonpanichakul T., Chan-In W., Opasawatchai A., Loison F., Matangkasombut O., Charoensawan V, & Matangkasombut P. (2021). Innate Lymphoid Cells Activation and Transcriptomic Changes in Response to Human Dengue Infection. Frontiers in Immunology, 12, 599805.

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Quantitative RT-PCR for THY-1 Expression

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Total RNA was extracted from HORCs using the RNeasy Mini Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. The concentration of total RNA was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). Total RNA was reverse transcribed to complementary DNA (cDNA) in a reaction mix of Superscript II reverse transcriptase (Invitrogen, Paisley, UK), dNTP mix (Bioline, London, UK) and random primers (Promega, Southampton, UK) according to manufacturer instructions.
TaqMan PCR was performed using 5ng of input cDNA and Taqman PCR mastermix (Applied Biosystems, Warrington, UK) and human THY-1 primer and probe set (Hs00174816_m1, Assay on demand, Applied Biosystems, Warrington, UK). Amplification and detection was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Warrington, UK). THY-1 mRNA was normalised to the geometric mean of C_T values for cytochrome c-1 (CYC-1) and topoisomerase DNA I (TOP1) as described previously [14 (link)]. Normalising genes were selected from a range of housekeeping genes using the Genorm protocol [17 (link)].

Osborne A., Aldarwesh A., Rhodes J.D., Broadway D.C., Everitt C, & Sanderson J. (2015). Hydrostatic Pressure Does Not Cause Detectable Changes in Survival of Human Retinal Ganglion Cells. PLoS ONE, 10(1), e0115591.

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Multiplex PCR for Genetic Profiling

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For this study, the following three PCR-based setups were carried out: M. leprae DNA screening (RLEP and 18-kDA), nuclear DNA SNP rs3135388 and mtDNA (hypervariable regions I and II) analysis. PCRs were performed in a 25-µL volume containing 1× Immolase buffer (Bioline), 0.04 U/µL Immolase DNA Polymerase (Bioline), 1.5 mM MgCl₂ (Bioline), 4% DMSO, 200 µM dNTP mix (Bioline) and 0.4 µM of each primer (see Supplementary Table 1 and Supplementary Fig. 1 for primer and amplicon details). For the M. leprae and the SNP screening, 5 µL of aDNA extract were used as template. For the mtDNA PCR, the amount of extract added was 1 µL. The annealing temperatures were 52 °C (M. leprae DNA screening), 58 °C (SNP PCR) or 60 °C (mtDNA screening), respectively. PCR success was evaluated by gel electrophoresis. Subsequent Sanger sequencing was performed following standard procedures.

Krause-Kyora B., Nutsua M., Boehme L., Pierini F., Pedersen D.D., Kornell S.C., Drichel D., Bonazzi M., Möbus L., Tarp P., Susat J., Bosse E., Willburger B., Schmidt A.H., Sauter J., Franke A., Wittig M., Caliebe A., Nothnagel M., Schreiber S., Boldsen J.L., Lenz T.L, & Nebel A. (2018). Ancient DNA study reveals HLA susceptibility locus for leprosy in medieval Europeans. Nature Communications, 9, 1569.

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Liver RNA Extraction and cDNA Synthesis

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Total RNA was isolated from liver samples using TRIzol (Invitrogen, Paisley, UK), according to the supplier's protocols. Purified RNA (2 μg) was then treated with DNase (DNA-free kit; Ambion, Austin, TX, USA) and used to generate first-strand cDNA with M-MLV Reverse Transcriptase (Invitrogen, Paisley, UK), utilizing random hexamers (Invitrogen, Paisley, UK) and dNTP mix (Bioline, London, UK), according to the manufacturer's protocol. The resultant cDNA was amplified with specific primers for mice in a total volume of 10 μL. Table 1 depicts the gene-specific primer sequences used in the study. Primer optimization and real-time quantitative PCR were performed according to Rincón-Cervera et al. [34 (link)].

Echeverría F., Valenzuela R., Bustamante A., Álvarez D., Ortiz M., Soto-Alarcon S.A., Muñoz P., Corbari A, & Videla L.A. (2018). Attenuation of High-Fat Diet-Induced Rat Liver Oxidative Stress and Steatosis by Combined Hydroxytyrosol- (HT-) Eicosapentaenoic Acid Supplementation Mainly Relies on HT. Oxidative Medicine and Cellular Longevity, 2018, 5109503.

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Quantitative Gene Expression Analysis

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Total RNA was isolated from liver samples using Trizol (Invitrogen, Paisley, United Kingdom), according to the supplier’s protocols. Purified RNA (2 μg) was then treated with DNasa (DNA free kit; Ambion, Austin, TX, USA) and used to generate first-strand cDNA with M-MLV reverse transcriptase (Invitrogen), using random hexamers (Invitrogen, Paisley, United Kingdom) and dNTP mix (Bioline, London, United Kingdom), according to the manufacturer`s protocol. The resultant cDNA was amplified with specific primer for mice in a total volume of 10 μL. Gene specific primer sequences used are shown in Additional file 1: Table S2. Primers were optimized to yield 95%-100% of reaction efficiency with PCR products by development in agarose gel to verify the correct amplification length. Real Time PCR was performed in a Strategen Mx3000P System (Agilent Technologies, California, USA) following the manufacturer`s recommendation (Applied Biosystems, Foster City, CA, USA). All the expression levels of the target genes under study were normalized by the expression of β-actin as internal control (Applied Biosystems, Foster City, CA, USA). Fold changes between groups was calculated by the 2(^-ΔΔCt) method.

Valenzuela R., Echeverria F., Ortiz M., Rincón-Cervera M.Á., Espinosa A., Hernandez-Rodas M.C., Illesca P., Valenzuela A, & Videla L.A. (2017). Hydroxytyrosol prevents reduction in liver activity of Δ-5 and Δ-6 desaturases, oxidative stress, and depletion in long chain polyunsaturated fatty acid content in different tissues of high-fat diet fed mice. Lipids in Health and Disease, 16, 64.

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Quantitative RT-PCR Analysis of Mouse Liver Transcripts

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Total RNA was extracted from ≤ 50 mg mouse liver tissue using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. For cDNA synthesis, 1 µg RNA was mixed with 50 ng Random Hexamer and 125 ng Oligo(dT)₁₈ primers, 2 µl of 5 × M-MLV RT buffer, 100 units of M-MLV Reverse Transcriptase, 10 units of RiboLock RNase Inhibitor (all from Thermo Scientific), 2 µl of 10 mM dNTP-Mix (Bioline), and RNase-free H₂O in 10 µl total volume. The mixture was then incubated at 25 °C for 10 min, 42 °C for 60 min for reverse transcription, and then 70 °C for 10 min to denature the enzyme in a thermal cycler (Applied Biosystems). cDNA was diluted 1:50 with DNase-free H2O for qPCR. Four microliters of diluted cDNA in combination with 5 µl 2X qPCRBIO SyGreen Mix Lo‐ROX (PCR Biosystems) and 0.5 µl of each forward and reverse primers at 10 µM concentration for the target genes (Supplementary Table. 1) was used for qPCR in a Rotor-Gene Q device (QIAGEN).

Zhou Z., Qian J., Kini A., Riederer B., Römermann D., Gros G, & Seidler U. (2022). Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice. Pflugers Archiv, 474(5), 529-539.

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Isolation and Characterization of Bone Marrow RNA

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Total RNA was isolated from extracted mononuclear cells (patient bone marrow samples). Total RNA was extracted using the E.Z.N.A^® Total RNA Kit I (Omega Bio-Tek Inc, Norcross, GA, USA) according to the manufacturer’s instructions. After isolation, RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized (with 1 μg of total RNA as a starting material) using SuperScript III Reverse Transcriptase (18080093, Invitrogen, Carlsbad, CA, USA), random primers (C1181, Promega, Madison, WI, USA), RiboLock(tm) Ribonuclease Inhibitor (#EO0381, Thermo scientific, Waltham, MA, USA) and dNTP-mix (BIO-39028, Bioline, London, UK). RT-reactions were performed according to the enzyme manufacturer’s instructions.

Mäkelä E., Löyttyniemi E., Salmenniemi U., Kauko O., Varila T., Kairisto V., Itälä-Remes M, & Westermarck J. (2019). Arpp19 Promotes Myc and Cip2a Expression and Associates with Patient Relapse in Acute Myeloid Leukemia. Cancers, 11(11), 1774.

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Transient Transfection of HEK293T Cells

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Human embryonic kidney (HEK293T) cells were transiently transfected in triplicate with 1.2 µg of OPN1LW-pMT4 recombinant expression vector using Attractene (Qiagen, Chadstone, VIC, Australia) in six-well cell culture plates. After 48 h, transfected cells were harvested using Trypsin-EDTA 1X (Sigma-Aldrich, Castle Hill, NSW, Australia), and washed four times with PBS (1X; 138 mM NaCl, 2.70 mM KCl, 10 mM NaPO₄, 1.8 mM KPO₄, pH 7.4). Total RNA was extracted using the PureLink RNA Mini with the TRIzol kit (Thermo Fisher Scientific, Scoresby, VIC, Australia), before the generation of oligo dT-primed cDNA using 5 µl (1–2 µg) of total RNA incubated with 5 µl oligo dT (500 ng) and 20.5 µl sterile RNase-free water for 15 min at 85 °C, followed by 2 min on ice. Subsequently, 10 µl of 5X First-Strand Buffer (Genesearch, Arundel, QLD, Australia), 5 µl (0.1 M) of DTT (Genesearch, Arundel, QLD, Australia), 2.5 µl (10 mM) dNTP mix (Bioline, Alexandria, NSW, Australia), and 1 µl RNase murine inhibitor (40 U/µl; Genesearch, Arundel, QLD, Australia) was added and incubated for 2 min at 25 °C. Then, 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for a further 5 min at 25 °C, followed by 1 h at 42 °C. Finally, a further 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for another 1 h at 42 °C.

Mountford J.K., Davies W.I., Griffiths L.R., Yazar S., Mackey D.A, & Hunt D.M. (2019). Differential stability of variant OPN1LW gene transcripts in myopic patients. Molecular Vision, 25, 183-193.

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Quantitative RT-PCR Analysis of Murine Retina

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Total RNA was isolated from murine retina and analysis was performed using an iQ5 Multicolor Real-time PCR I-cycler (Bio-Rad Laboratories, Hercules, CA) as described previously [15 (link)]. Reactions were performed with cDNA (6 ng/reaction, in triplicate) using 1X PCR buffer (Bioline), 3 mM MgCl₂, 10 nM fluorescein (USB Corporation), 0.1% Triton X-100, 0.03 U/μL Immolase DNA polymerase (Bioline), 800 μM dNTP mix (Bioline), 0.025X SYBR Green (Invitrogen), and 200 nM of forward and reverse primers in a total volume of 25 μL. Normalized gene expression was determined using the iQ5 optical system software (BioRad) using acidic ribosomal phosphoprotein P0 (ARBP) expression as a reference gene for each sample. The sequence for primers used in qRT-PCR is provided in Table 2.

Schuld N.J., Hussong S.A., Kapphahn R.J., Lehmann U., Roehrich H., Rageh A.A., Heuss N.D., Bratten W., Gregerson D.S, & Ferrington D.A. (2015). Immunoproteasome Deficiency Protects in the Retina after Optic Nerve Crush. PLoS ONE, 10(5), e0126768.

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