TRAIL- and PTEN-bearing mRNAs were generated by in vitro transcription. The human 5′UTR with Kozak sequence and 3′UTR sequence were synthesized commercially by Integrated DNA Technologies (Coralville, Iowa) and sub-cloned into pcDNA3.3. Plasmid inserts were excised by restriction enzyme digestion and used to template tail PCRs. The templates of human TRAIL and PTEN were obtained from our previously constructed expression vectors [19 (link), 47 (link)]. MEGAscript T7 kit (Ambion) was used to synthesize mRNA. However, m7GpppG was replaced with ARCA cap analog (New England Biolabs) and cytidine and uridine were replaced with 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies) respectively. Reactions were incubated 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5′-triphosphates. The synthesized RNA was purified with Ambion MEGAclear spin columns (Ambion) and quantitated by Nanodrop (Thermo Scientific).
Ambion megaclear spin columns
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion MEGAclear spin columns are designed for the purification of RNA from cell culture, tissue, or other sample types. The columns utilize a silica-based membrane to capture RNA, allowing for efficient removal of salts, enzymes, and other contaminants. The purified RNA can then be eluted from the column for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Antarctic phosphatase, by New England Biolabs (6 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (5 mentions)
Arca cap analog, by New England Biolabs (4 mentions)
5 methylcytidine triphosphate, by TriLink (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Pseudouridine triphosphate, by TriLink (3 mentions)
Transit mrna, by Mirus Bio (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Beyotime (1 mentions)
Pcdna 3.4 topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
N1 methyl pseudouridine, by TriLink (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using ambion megaclear spin columns
1
In vitro Synthesis of Capped and Modified mRNA
2
Synthetic mRNA Transfection in Cells
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ORFs encoding TGIF2, PDX1, NeuroD1, and Mafa were cloned from mouse islet cDNA by polymerase chain reaction (PCR). In vitro transcribed template construction and RNA synthesis are schematized in Figure 1A . mRNAs were synthesized with the use of the MEGAscript T7 kit (Ambion) according to the manufacturer’s instructions. The reaction used ARCA cap analog (New England Biolabs), 5-methylcytidine triphosphate instead of cytidine and pseudouridine triphosphate instead of uridine (TriLink Biotechnologies). Reactions were incubated for 5 h at 37°C followed by Antarctic Phosphatase (New England Biolabs) treatment for 2 h at 37°C to remove residual triphosphates. The synthesized RNA was purified with Ambion MEGAclear spin columns (Ambion) and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific). mRNA transfection was carried out with TransIT-mRNA (Mirus). In vitro transcribed mRNAs were diluted in Opti-MEM basal media (Gibco), followed by the addition of boost reagent and TransIT-mRNA sequentially. After 2 min incubation at room temperature (RT), the RNA–lipid complexes were delivered to the culture medium. Four hours later, the medium was replaced with normal culture medium.
3
In Vitro Synthesis of Therapeutic mRNAs
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Luc-mRNA, RFP-mRNA, and TRAIL-mRNAs were synthesized in vitro as previously described.32 (link) Briefly, the human 5′ UTR with Kozak sequence and 3′ UTR sequence were commercially synthesized by Integrated DNA Technologies (Coralville, IA) and sub-cloned into pcDNA3.3. The DNA templates of human TRAIL and luciferase were obtained from our previously constructed expression vectors through restriction enzyme digestion. MEGAscript T7 kit (Ambion) was used to synthesize mRNAs, whereas m7GpppG was replaced with ARCA cap analog (New England Biolabs) and cytidine and uridine were replaced with 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies) respectively. Reactions were sustained for 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5′-triphosphates. The synthesized mRNAs were purified with Ambion MEGAclear spin columns (Ambion) and quantitated with Nanodrop (Thermo Fisher Scientific).
4
In Vitro Synthesis of mRNA Reporters
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In vitro synthesis of Luc-mRNA and TRAIL-mRNA Luc-mRNA and TRAIL-mRNAs were synthesized in vitro as previously described [6] . Brie y, the human 5'UTR with Kozak sequence and 3'UTR sequence were commercially synthesized by Integrated DNA Technologies (Coralville, Iowa) and sub-cloned into pcDNA3.3. The DNA templates of human TRAIL and luciferase were obtained from our previously constructed expression vectors through restriction enzyme digestion. MEGAscript T7 kit (Ambion) was used to synthesize mRNAs, whereas m7GpppG was replaced with ARCA cap analog (New England Biolabs) and cytidine and uridine were replaced with 5methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies) respectively.
Reactions were sustained for 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5'-triphosphates. The synthesized mRNAs were puri ed with Ambion MEGAclear spin columns (Ambion) and quantitated with Nanodrop (Thermo Scienti c).
Reactions were sustained for 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5'-triphosphates. The synthesized mRNAs were puri ed with Ambion MEGAclear spin columns (Ambion) and quantitated with Nanodrop (Thermo Scienti c).
5
Cloning and Expression of Human TRAIL in ADSCs
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Full-length human TRAIL cDNA was cloned into the pcDNA3.3-TOPO plasmid using a pcDNA 3.4 TOPO TA Cloning Kit (#A14697; Thermo Fisher Scientific, Inc.) as shown in Fig. 1A . In brief, TRAIL cDNA was amplified from MIGR1-TRAIL-GFP as described by Wiley et al (18 (link)) and then sub-cloned into the pcDNA3.3-TOPO plasmid. The TRAIL cDNA was connected to the pcDNA 3.3-TOPO plasmid with T4 DNA ligase (#D7006; Beyotime Institute of Biotechnology, Beijing, China). After colony polymerase chain reaction (PCR) amplification (the template was bacterium suspension) and DNA sequencing confirmation (performed by Sangon Biotech Co., Ltd., Wuhan, China), this plasmid containing TRAIL cDNA was used for tail PCR and modified with the MEGAscript T7 kit (Ambion; Thermo Fisher Scientific, Inc.). Then, modified TRAIL mRNA was isolated with Ambion Anti-Reverse Cap Analog (ARCA; #AM8045) and purified with Ambion MEGAclear spin columns (Thermo Fisher Scientific, Inc.) and treated with Antarctic Phosphatase (New England Biolabs, Ipswich, MA, USA) to remove residual 5′-triphosphates. The transfection of the TRAIL plasmid into ADSCs was conducted using TransIT-mRNA (Mirus Bio LLC., Madison, WI, USA) according to the manufacturer' instructions and ADSCs transfected with TRAIL-cDNA were defined as TRAIL-ADSCs.
6
In Vitro Synthesis of Modified mRNA with C-terminal Tags
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Modified mRNA of monomeric or bivalent VHHs, including a range of C-terminal tags: hemagglutinin (HA) tag, HA-mCherry, HA-mCherry-PEST or T2A-mCherry, were synthesised in vitro using T7 RNA polymerase-mediated transcription from a linearised synthetic DNA template, which incorporates generic 5′ and 3′ UTRs and a poly-A tail, as previously described68 (link),69 (link) using Megascript T7 transcription kit (Thermo Fisher Scientific, SE). For all these RNAs, uridine was fully replaced by N1-methylpseudouridine (Trilink, USA) in the in vitro transcription reaction. The RNA was purified using Ambion MEGA clear spin columns (Thermo Fisher Scientific, SE) and treated with Antarctic Phosphatase (New England Biolabs, SE) for 30 min at 37 °C to remove residual 5′-phosphates. The RNA was then re-purified and quantified by Nanodrop (Thermo Fisher Scientific, SE). After purification, modRNA was resuspended in 10 mM Tris HCl, 1 mM EDTA at 1 μg/μl for use in in vitro experiments. For the use of in vivo experiments, large-scale batches of modRNA (400 μg) were concentrated to 10 μg/μl using ethanol precipitation.
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