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).
Arca cap analog
Manufactured by New England Biolabs
Sourced in United States
The ARCA cap analog is a chemical compound used in in vitro RNA transcription reactions to synthesize capped RNA transcripts. It functions as a structural mimic of the 5' cap structure found on eukaryotic mRNA, facilitating efficient translation initiation.
Automatically generated - may contain errors
Lab products found in correlation
Megascript t7 kit, by Thermo Fisher Scientific (5 mentions)
Nanodrop, by Thermo Fisher Scientific (5 mentions)
5 methylcytidine triphosphate, by TriLink (4 mentions)
Ambion megaclear spin columns, by Thermo Fisher Scientific (4 mentions)
Antarctic phosphatase, by New England Biolabs (4 mentions)
Pseudouridine triphosphate, by TriLink (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
B18r interferon inhibitor, by Thermo Fisher Scientific (1 mentions)
Kapa taq kit, by Roche (1 mentions)
Pseudo utp, by TriLink (1 mentions)
5 methyl ctp, by TriLink (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Transit mrna, by Mirus Bio (1 mentions)
5 protocols using arca cap analog
1
In vitro Synthesis of Capped and Modified mRNA
2
In Vitro mRNA Synthesis and Transfection in hESCs
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In vitro mRNA synthesis and transfections were performed according to previous protocols.46 (link) In brief, T7 promoter and polyA tail were added by polymerase chain reaction (PCR) using KAPA taq kit (Kapabiosystems, London, UK). RNA was transcribed from the template using MEGAscript T7 kit (Ambion, Carlsbad, CA, USA), with ARCA cap analog (New England Biolabs, Ipswich, MA, USA); ATP; GTP; 5-Methyl-CTP (TriLink, San Diego, CA, USA); and pseudo-UTP (TriLink). Synthesized RNAs were purified with the MEGAclear kit (Ambion). RNA transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To increase the viability of transfected cells, B18R interferon inhibitor (eBioscience, San Diego, CA, USA) was supplemented to the culture medium. One day before transfection, 30,000 hESCs were seeded on a culture plate, and 1 µg/well of each synthetic modified mRNA was induced. The hESCs were subjected to four consecutive transfections with TF-encoding RNAs, or GFP or mCherry mRNAs as controls with Lipofectamine 2000 (Life Technologies) in StemFit AK-03 with B18R (eBioscience) for the first two days of differentiation. After two consecutive days of transfection, the culture medium was changed to DKSFM with 100 μg/mL CT and 10 ng/mL EGF.
3
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.
4
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).
5
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).
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