Human tissue biobank cDNA (Primerdesign Ltd) was used as a template for the identification of ABC transporters in human tissue of interest. Equine peri-renal adipose tissue collection from clinical cases euthanized for reasons other than endocrine disease/systemic inflammation was approved by the University of Edinburgh Veterinary ethics and research committee. Where applicable, total RNA extraction was carried out by centrifugation in Qiazol lysis reagent using an RNeasy mini kit (Qiagen), according to the manufacturer's instructions. RNA integrity was checked on a 1% agarose gel, after which cDNA (500 ng RNA/reaction) was synthesized using a high capacity cDNA kit (Invitrogen), according to the manufacturer's instructions. cDNA was used as a template for specific primers (Invitrogen; table S4) for RT-PCR and amplified using GoTaq DNA polymerase (Promega) as per the manufacturer's instructions, on a Techne thermocycler (95 C for 5 min; 35 cycles of 95 C for 30 s, 60 C for 30 s, 72 C for 30 s; 72 C for 5 min). Products were subjected to gel electrophoresis on a 2% agarose gel in TAE buffer (50x stock buffer: 2 M Tris Base, 1 M glacial acetic acid, 100 mM disodium EDTA) containing GelRed (Cambridge Bioscience). Gels were imaged using a Gel-Doc system (Uvitec). Product size was confirmed against a 100 bp DNA ladder (Invitrogen).
Gelred
Manufactured by Cambridge Bioscience
Sourced in United Kingdom
GelRed is a fluorescent nucleic acid stain used for the detection of DNA or RNA in agarose gels. It is designed to be simple, safe, and effective for visualizing nucleic acids in electrophoresis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Micro bio spin column, by Bio-Rad (2 mentions)
Ni nta agarose beads, by Thermo Fisher Scientific (2 mentions)
100 kda filter, by Merck Group (2 mentions)
Purexpress cell free transcription translation kit, by New England Biolabs (2 mentions)
Amicon ultracel 0.5ml spin concentrator, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Gel doc system, by Uvitec (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Primetime qpcr probe, by Integrated DNA Technologies (1 mentions)
Sidestep lysis and stabilization buffer, by Agilent Technologies (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Biomix red pcr reaction mix, by Meridian Bioscience (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Red hot dna polymerase, by Thermo Fisher Scientific (1 mentions)
Biotaq dna polymerase, by Meridian Bioscience (1 mentions)
7 protocols using gelred
1
Identifying ABC Transporters in Human Tissues
2
Amplification and Sequencing of VGSC and nad4 Genes
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The primers used to amplify exons 21 and 31 of the VGSC gene, which encode for domain II subunit 5 and domain III subunit 6, respectively [31 (link)], as well as the mitochondrial nad4 gene [35 (link)] are described in Additional file 1 : Table S2. Polymerase chain reaction (PCR) was carried out in a total volume of 25 µl using standard protocols with 1× reaction buffer, 200 µM dNTP, 0.5 µM forward primer, 0.5 µM reverse primer, 0.02 U/µl DNA polymerase, 17.5 µl water and 1.5 µl DNA sample (1–5 ng). The PCR thermocycler profile consisted of: 1 cycle at 98 °C for 30 s, 30 cycles at 98 °C for 10 s, 56 °C (VGSC gene) or 59 °C (nad4 gene) for 30 s, 72 °C for 30 s and a final cycle at 72 °C for 2 min. PCR products (10 µl each) were detected by 1% agarose gel electrophoresis in TAE buffer, stained using GelRed (Cambridge Bioscience, Cambridge, UK). The samples were sequenced by capillary sequencing. The resulting DNA sequences have been submitted to GenBank under the accession numbers MT721877-MT721961.
3
Optimized RNA Extraction and qRT-PCR Analysis
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RNA was extracted using commercial kits (GenElute Mammalian Total RNA Miniprep Kit; RNeasy Mini Kit, Qiagen, Manchester, UK; SideStep Lysis and Stabilization Buffer, Agilent Technologies, Wokingham, UK) and reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Life Technologies, Paisley, UK). Oligonucleotides and PrimeTime qPCR probes (Integrated DNA Technologies, Glasgow, UK) used for real-time RT-PCR are shown in Table 1 . TaqMan assays for RNA polymerase II (RNAP) (POLR2A Hs00172187_m1) and Collagen Type 6A3 (COL6A3 Hs00915125_m1) were from Life Technologies. For real-time RT-PCR, relative gene expression levels were determined in cDNA samples using TaqMan Universal Master Mix (Life Technologies) and the ABI Prism 7900HT sequence detection system (Life Technologies). Expression levels were normalised to RNAP. Primers used for conventional real-time RT-PCR are shown in Table 2 . For RT-PCR, cDNA was amplified using Bio-Mix Red PCR reaction mix (Bioline, London, UK) and analysed on 3.5% agarose gels stained with Gel-Red (Biotium, Cambridge Bioscience, Cambridge, UK) using β2-microglobulin (β2m) or 18S rRNA as a reference gene.
4
In Vitro Synthesis of ShdA Domains
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In vitro synthesis of the RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV was performed using the PURExpress cell-free transcription/translation kit (NEB). Protein synthesis was performed with either 250 ng and 500 ng of DNA template. Synthesis was performed for 4 hr at 37°C according to the manufacturer’s recommendation. Following incubation, 10 mM MgCl2 was added to the reaction and the final volume was adjusted to 10 μL. Ribosomes were removed through centrifugation for 60 min at 15,000 rpm at 4°C, through an Amicon Ultracel 0.5mL spin concentrator with a 100 KDa filter (Merck). The flowthrough was collected and the His-tagged PURExpress kit components were removed from the reaction following incubation with Ni-NTA agarose beads (Thermo) for 45 min at 4°C. Agarose beads were removed through centrifugation with Biorad micro Bio-spin columns at 15,000 g for 10 min at 4°C. As a control, the same reactions were performed in parallel with the dihydrofolate reductase (DHFR) control provided by the PURExpress kit.
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).
5
In Vitro Synthesis of ShdA Domains
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In vitro synthesis of the RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV was performed using the PURExpress cell-free transcription/translation kit (NEB). Protein synthesis was performed with either 250 ng and 500 ng of DNA template. Synthesis was performed for 4 hr at 37°C according to the manufacturer’s recommendation. Following incubation, 10 mM MgCl2 was added to the reaction and the final volume was adjusted to 10 μL. Ribosomes were removed through centrifugation for 60 min at 15,000 rpm at 4°C, through an Amicon Ultracel 0.5mL spin concentrator with a 100 KDa filter (Merck). The flowthrough was collected and the His-tagged PURExpress kit components were removed from the reaction following incubation with Ni-NTA agarose beads (Thermo) for 45 min at 4°C. Agarose beads were removed through centrifugation with Biorad micro Bio-spin columns at 15,000 g for 10 min at 4°C. As a control, the same reactions were performed in parallel with the dihydrofolate reductase (DHFR) control provided by the PURExpress kit.
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).
6
RNA Extraction and cDNA Synthesis Protocol
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RNA was extracted with Direct-zol™ RNA MiniPrep Kit (Zymo Research, California). cDNA was synthesised from 1 µg RNA with Moloney Murine Leukaemia Virus Reverse Transcriptase (USB) in a 20 μl reaction and 0.1 μl cDNA amplified with Red Hot DNA polymerase (Thermo-scientific) in 10 μl [26 (link), 27 (link)]. Primers designed with Primer-BLAST were synthesised by SIGMA-Aldrich. Amplicons were analysed on 3% agarose gels, stained with GelRed (Cambridge Bioscience, Cambridge, UK).
7
DNA Amplification and Sequencing
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PCR was carried out with BioTaq DNA polymerase according to standard protocol (Bioline) and thermocycling consisting of 30 cycles of 95 with GelRed (Cambridge Bioscience) and a UVP documentation system. Any bands in addition to the predicted amplicon on the gel were isolated using the band stab method of Bjourson and Cooper, 1992 [15] . Re-amplification of these isolated sequences was performed by PCR as described above. DNA sequencing of isolated PCR products was performed as described previously [11] .
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