Custom designed primers

Manufactured by Integrated DNA Technologies

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Custom-designed primers are short, synthetic DNA sequences that serve as starting points for DNA amplification in various molecular biology applications. They are designed to target specific regions of a DNA template and facilitate the initiation of DNA synthesis by DNA polymerase enzymes.

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Custom primers (17 citations) Primers designed (2 citations)

7 protocols using custom designed primers

Gene Expression Analysis in Mouse Kidney and Human Cells

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RNA was isolated from mouse whole-kidney tissue and HK-2 cells using TRIzol reagent (Thermo Fisher Scientific), and cDNA was reverse transcribed from 0.2 μg (mouse kidney) and 1 μg (HK-2 cells) RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Custom-designed primers were from Integrated DNA technologies (Coralville, IA, USA) and had the following sequences: mouse Ccr2 (forward, 5′-AAAGGAGCCATACCTGTAAATGCC-3′; reverse, 5′-TGCCGTGGATGAACTGAGGTAAC-3′), mouse Col1a1 (forward, 5′-TTCAGGGAATGCCTGGTGAA-3′; reverse, 5′-ACCTTTGGGACCAGCATCA-3′), mouse Col1a2 (forward, 5′-GAAAAGGGTCCCTCTGGAGAA-3′; reverse, 5′-AATACCGGGAGCACCAAGAA-3′), mouse Rpl13a (forward, 5′-GCTCTCAAGGTTGTTCGGCTGA-3′; reverse, 5′-AGATCTGCTTCTTCTTCCGATA-3′), human PPARGC1A (forward, 5′-AGCCTCTTTGCCCAGATCTT-3′; reverse, 5′-GGCAATCCGTCTTCATCCAC-3′), and human RPL13A (forward, 5′-TCGTACGCTGTGAAGGCATC-3′; reverse, 5′-TTTTGTGGGGCAGCATACCT-3′). Primers for human PPARA (HP226273), PPARD (HP209214), PPARG (HP226175) and CCN2 (HP205671) were from OriGene (Rockville, MD, USA).

Hong L.Y., Yeung E.S., Tran D.T., Yerra V.G., Kaur H., Kabir M.D., Advani S.L., Liu Y., Batchu S.N, & Advani A. (2023). Altered expression, but small contribution, of the histone demethylase KDM6A in obstructive uropathy in mice. Disease Models & Mechanisms, 16(9), dmm049991.

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Platelet mRNA Quantification Protocol

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Full details of mRNA analysis of platelet populations are available in supplemental Methods. RNA was extracted from fluorescence-activated cell-sorted platelets (2.5 million per population) using the miRNeasy mini kit (Qiagen, 217004), following the manufacturer’s instructions. Samples were spiked with Cel-miR-39-3p (Qiagen, 219600) and MS2 carrier RNA (Roche, 10165948001) after the first step of RNA isolation (ie, addition of Qiazol to sample). Complementary DNA was generated using VILO RT Superscript (Thermo Fisher, 11755-250) from 8 μL of RNA, as per the manufacturer’s instructions. Transcripts were quantified using SYBR Select Master Mix (Applied Biosystems, 4472908) with custom-designed primers (Integrated DNA Technologies), in a ViiA 7 real-time polymerase chain reaction system (Applied Biosystems) using ViiA 7 software (Applied Biosystems).

Armstrong P.C., Allan H.E., Kirkby N.S., Gutmann C., Joshi A., Crescente M., Mitchell J.A., Mayr M, & Warner T.D. (2022). Temporal in vivo platelet labeling in mice reveals age-dependent receptor expression and conservation of specific mRNAs. Blood Advances, 6(23), 6028-6038.

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Plasmid and Genomic DNA Isolation

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Plasmid DNA was isolated from E. coli cells using a commercial kit (Qiagen). A. baumannii total DNA was isolated using a phenol-based method described previously [21 (link)]. DNA polymerases and restriction enzymes were used according to the manufacturer’s protocols (New England Biolabs). Custom-designed primers (Integrated DNA Technologies) or kit-supplied M13 or T7 primers (Life Technologies) were used to perform all sequencing reactions using BigDye-based chemistry (Applied Biosystems) prior to subcloning reactions and DNA transformations. All primers used in this work are listed in S1 Table.

Wood C.R., Squire M.S., Finley N.L., Page R.C, & Actis L.A. (2019). Structural and functional analysis of the Acinetobacter baumannii BlsA photoreceptor and regulatory protein. PLoS ONE, 14(8), e0220918.

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Chondrogenic Gene Expression Analysis

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Lipofectamine 3000 (Thermo Fisher Scientific) was used to transfect mouse chondrogenic ATDC5 cells (42 (link)) with various combinations of expression plasmids for mouse SOX5, mouse SOX6, and human full-length or mutant SOX9. Total RNA was prepared 24 h later using TRIzol (Life Technologies) and following manufacturer's instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qPCR was performed using the StepOne Plus Real Time PCR system (Thermo Fisher Scientific), SYBR Green PCR Master Mix (Thermo Fisher Scientific) and custom-designed primers (Integrated DNA Technologies) (Supplementary Table S5). Col2a1 and Acan mRNA levels were calculated relative to those of Hprt according to the ΔΔCt method.

Haseeb A, & Lefebvre V. (2019). The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism. Nucleic Acids Research, 47(13), 6917-6931.

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Cytokine Expression Analysis in Lung Tissue

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Whole lungs were collected and stored in RNA Stabilization Reagent, RNAlater (Qiagen, Chadstone Centre, Vic, Australia). RNA was extracted with guanidinium thiocyanate phenol chloroform (TRIzol). Extracted RNA was treated with DNAse I (Sigma, Castle Hill, NSW, Australia) and reverse‐transcribed using Bioscript (Bioline, Alexandria, NSW, Australia) and random hexamer primers (Invitrogen, Mount Waverley, Vic, Australia).21, 59, 79 The relative abundance of cytokine transcripts for IL‐6, TNF‐α, CXCL1, CXCL2, fibronectin (Fn) and matrix metalloproteinase‐12 (MMP‐12) was determined relative to the reference gene hypoxanthine phosphoribosyltransferase (HPRT) by real‐time qPCR using a ViiA 7 Real‐Time PCR System (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA). Custom‐designed primers (Integrated DNA Technologies, Coralville, Iowa, USA) were used (Supplementary table 1).

Nair P.M., Starkey M.R., Haw T.J., Liu G., Collison A.M., Mattes J., Wark P.A., Morris J.C., Verrills N.M., Clark A.R., Ammit A.J, & Hansbro P.M. (2019). Enhancing tristetraprolin activity reduces the severity of cigarette smoke‐induced experimental chronic obstructive pulmonary disease. Clinical & Translational Immunology, 8(10), e01084.

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Gene Rearrangement Confirmation via PCR

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Confirmation of the gene rearrangement identified by MPseq was sought using PCR amplification with TaqMan PCR Master Mix (ThermoFisher Scientific) with custom designed primers (Integrated DNA Technologies), followed by amplicon clean up with ExoSAP-IT Express (ThermoFisher Scientific) and dye terminator sequencing up to 800 bp using the BigDye Terminator v1.1 chemistry (Applied Biosystems International) on the ABI 3730xl DNA Analyzer instrument.

Venable E.R., Gagnon M.F., Pitel B.A., Palmer J.M., Peterson J.F., Baughn L.B., Hoppman N.L., Greipp P.T., Ketterling R.P., Patnaik M.S., Kelemen K, & Xu X. (2023). A TRIP11:: FLT3 gene fusion in a patient with myeloid/lymphoid neoplasm with eosinophilia and tyrosine kinase gene fusions: a case report and review of the literature. Cold Spring Harbor Molecular Case Studies, 9(1), a006243.

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Quantitative Real-Time PCR Analysis

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Real-time qRT-PCR for genes PTEN, GAPDH, GUSB, COL11A2, and SAMD11 was performed using TaqMan Gene Expression Assays (Applied Biosystems), listed in Table S4. qRT-PCR for genes RAB11FIP1, ATP23, MFSD6, PRCD, CDYL, COX17, and FOXD1 was performed using QuantiFast SYBR Green PCR Master Mix (QIAGEN) and custom-designed primers (Integrated DNA Technologies), listed in Table S5. qRT-PCR reactions were carried out in the ViiA 7 Real-Time PCR System (Applied Biosystems). TaqMan assays were performed with the following thermocycling settings: 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. SYBR Green assays were performed with the following thermocycling settings: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Cycle threshold (Ct) was automatically determined for each well using QuantStudio Real Time PCR Software (version [v.]1.1, Applied Biosystems). Relative quantification of gene expression was carried out according to the comparative (ΔΔ) Ct method,101 (link), 102 (link) with GAPDH and GUSB as housekeeping genes.

Moses C., Nugent F., Waryah C.B., Garcia-Bloj B., Harvey A.R, & Blancafort P. (2018). Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. Molecular Therapy. Nucleic Acids, 14, 287-300.

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