Clean PCR products generated with plasmid templates, purchased from GenScript, were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of ARCA; 3′-O-Me-m7G(5′)ppp(5′)G (Trilink Biotechnologies, San Diego, CA, USA); Guanosine-5′-triphosphate (GTP); Adenosine triphosphate (ATP); Cytidine triphosphate (CTP); Uridine-5′-triphosphate (UTP) (in case of synthetic mRNA) (Life Technologies, Carlsbad, CA, USA); and N1-Methylpseudouridine-5′-triphosphate (Trilink Biotechnologies) in the case of synthetic modRNA [24 (link)]. The mRNA was purified either with the MEGA clear kit (Life Technologies) according to the manufacturer’s instructions or using Amicon Ultra-4 Centrifugal Filter Unit 4 mL,10 kDa (Millipore Sigma, Burlington, MA, USA) and treated with Antarctic Phosphatase (NEB). Next, the mRNA was re-purified with the MEGA clear kit. The mRNA was quantified using a Nano Drop spectrometer (Thermo Scientific, Waltham, MA, USA), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM Tris-HCl and 1 mM EDTA. The open reading frame for the Luc and nGFP modRNA is listed below. Endogenous Luc mRNA is a mixture of mRNA enriched with luciferase gene, obtained by isolation of RNA form 4T1-Luc cell line (ATCC #CRL2539LUC2).
Cytidine triphosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cytidine triphosphate (CTP) is a nucleotide that serves as a building block for ribonucleic acid (RNA) synthesis. It is one of the four primary nucleotides found in RNA, along with adenosine triphosphate (ATP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). CTP is an essential component in the cellular processes that generate RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Thermo Fisher Scientific (8 mentions)
Megaclear kit, by Thermo Fisher Scientific (8 mentions)
Guanosine triphosphate, by Thermo Fisher Scientific (6 mentions)
Antarctic phosphatase, by New England Biolabs (6 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
3 o me m7g 5 ppp 5 g, by TriLink (5 mentions)
N1 methylpseudouridine 5 triphosphate, by TriLink (5 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (5 mentions)
Megaclear kit, by New England Biolabs (4 mentions)
Antarctic phosphatase, by Merck Group (2 mentions)
Geneart, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Geneart gene synthesis, by Thermo Fisher Scientific (1 mentions)
8 protocols using cytidine triphosphate
1
Synthetic mRNA Production and Purification
2
Synthesis and Purification of Modified RNA
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ModRNAs were synthesized in house. All DNA plasmid templates used for modRNA production were purchased from GeneArt and Invitrogen and were verified by sequencing. PCR-tailed DNA plasmids (120 poly(A) tail) were used for in vitro transcription to generate modRNAs (see Table S1 for complete list of open reading frame sequences used for modRNA synthesis in this study) with a customized ribonucleotide blend of anti-reverse cap analog, 3′-O-Me-m7G(5′)ppp(5′)G (6 mM, TriLink Biotechnologies), guanosine triphosphate (1.5 mM, Life Technologies), adenosine triphosphate (7.5 mM, Life Technologies), cytidine triphosphate (7.5 mM, Life Technologies), and 1-mψU (7.5 mM, TriLink Biotechnologies), as previously described.37 (link), 38 (link), 39 (link), 40 (link) We purified the mRNA with the Megaclear kit (Life Technologies) and treated it with Antarctic phosphatase (New England Biolabs) for 1 hr, before repurification with the Megaclear kit. The mRNA was precipitated by incubation overnight with ammonium acetate and ethanol at 4°C and resuspended in 10 mM TrisHCl, 1 mM EDTA. We quantified the mRNA with a Nanodrop spectrometer (Thermo Scientific) and checked its quality with a bioanalyzer (Agilent Technologies). A more detailed description of the protocol for modRNA synthesis is provided elsewhere.39 (link)
3
Efficient Synthesis of Customized modRNA
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All modRNA was generated our laboratory by in vitro transcription of plasmid templates (GeneArt, Thermo Fisher Scientific). The full list of open reading frame sequences used to make modRNA for this study can be found in Table S1 . The transcription step involved a customized ribonucleotide blend of anti-reverse cap analog; 30-O-Me-m7G(50) ppp(50)G (6 mM, TriLink Biotechnologies); guanosine triphosphate (1.5 mM, Life Technologies); adenosine triphosphate (7.5 mM, Life Technologies); cytidine triphosphate (7.5 mM, Life Technologies) and N1-methylpseudouridine-5-triphosphate (7.5 mM, TriLink Biotechnologies). Next, modRNA was purified with the Megaclear kit (Life Technologies) and treated with Antarctic Phosphatase (New England Biolabs). To eliminate any remaining impurities, modRNA was re-purified with the Megaclear kit and quantified using a Nanodrop spectrometer (Thermo Scientific). Lastly, modRNA was precipitated with ethanol and ammonium acetate and resuspended in 10 mM Tris-HCl and 1 mM EDTA.
4
mRNA Generation via In Vitro Transcription
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In brief, clean PCR products generated with plasmid templates purchased from GenScript were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of ARCA; 30-O-Me-m7G(50) ppp(50)G (catalog no. [cat. #] N-1081; Trilink Biotechnologies); GTP (cat. #am1334-5; Life Technologies); ATP (cat. #am1334-5; Life Technologies); cytidine triphosphate (cat. #am1334-5; Life Technologies), and N1-methylpseudouridine-50-triphosphate (cat. #N-1081; Trilink Biotechnologies). The mRNA was purified with the MEGAclear kit (cat. #AM1908; Life Technologies) according to the manufacturer’s instructions or using Amicon Ultra-4 Centrifugal Filter Unit 4 mL,10 kDa (cat. #UFC801024; MilliporeSigma) and treated with Antarctic Phosphatase (cat. #M0289L; NEB). It was then re-purified with the MEGAclear kit. The mRNA was quantified using a NanoDrop spectrometer (Thermo Scientific), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM Tris-HCl and 1 mM EDTA. The open reading frame for the modRNA used is listed in Table S1 . This protocol has been described in detail elsewhere.29 (link)
5
In Vitro Synthesis of Modified mRNA
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Clean PCR products generated with plasmid templates (GeneArt; Thermo Fisher Scientific) were used as the template for mRNA. modRNAs were generated by transcription in vitro with a customized ribonucleoside blend of anti-reverse cap analog, 3′-O-Me-m7G(5′)ppp(5′)G (6 mM; TriLink Biotechnologies), guanosine triphosphate (1.5 mM; Life Technologies), adenosine triphosphate (7.5 mM; Life Technologies), cytidine triphosphate (7.5 mM; Life Technologies), and N1-Methyl-Pseudouridine-5'-Triphosphate (7.5 mM; TriLink Biotechnologies). The mRNA was purified with the MEGAclear kit (Life Technologies) and treated with Antarctic Phosphatase (New England Biolabs). It was then repurified with the MEGAclear kit. The mRNA was quantified on a NanoDrop spectrometer (Thermo Scientific), precipitated with ethanol and ammonium acetate, and resuspended in 10 mM Tris-HCl and 1 mM EDTA.
6
In Vitro Synthesis of Modified mRNA
7
In vitro Transcription of ModRNA
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ModRNAs were transcribed in vitro from plasmid templates (see Table S1 (Supporting Information) for a complete list of open reading frame sequences used to make the modRNA for this study) using a customized ribonucleotide blend of anti‐reverse cap analog; 3 ´‐O‐Me‐m7G (5')ppp(5')G (6 × 10−3m , TriLink Biotechnologies); guanosine triphosphate (1.5 × 10−3m , Life Technology); adenosine triphosphate (7.5 × 10−3m , Life Technology); cytidine triphosphate (7.5 × 10−3m , Life Technology); and N1‐Methylpseudouridine‐5'‐Triphosphate (7.5 × 10−3m , TriLink Biotechnologies) as described previously in the recent protocol paper.[52 ] The mRNA was purified using the Megaclear kit (Life Technology) and treated with antarctic phosphatase (New England Biolabs), followed by re‐purification using the Megaclear kit. The mRNA was quantitated by Nanodrop (Thermo Scientific), precipitated with ethanol and ammonium acetate, and resuspended in 10 × 10−3m TrisHCl, 1 × 10−3m EDTA.
8
In Vitro Transcription of Modified RNA
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DNA plasmids for RIPK3WT, RIPK3S204>A, RIPK3S232>A or RIPK3DM were purchased from GeneArt Gene Synthesis (Thermo Fisher). In vitro transcription was made using clean tailed (120- poly A tail) DNA PCR products. ModRNAs were transcribed in vitro using a custom ribonucleoside blend of Anti Reverse Cap Analog, 30-O-Me-m7G(50) ppp(50)G (6 mM, TriLink Biotechnologies), guanosine triphosphate (1.5 mM, Life Technologies), adenosine triphosphate (7.5 mM, Life Technologies), cytidine triphosphate (7.5 mM, Life Technologies), N1-Methylpseu-douridine-50-Triphosphate (7.5 mM, TriLink Biotechnologies). The mRNA was purified using a Megaclear kit (Life Technologies) and was treated with Antarctic Phosphatase (New England Biolabs); then it was purified again using the Megaclear kit. The mRNA was quantitated by Nanodrop (Thermo Scientific), precipitated with ethanol and ammonium acetate, and re-suspended in 10 mM TrisHCl and 1 mM EDTA70 (link).
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