Sep 16, 2020

Public workspacemRNA Synthesis and Transfection

This protocol is a draft, published without a DOI.
  • Yingchao Xue1,2,
  • Xiping Zhan3,
  • Shisheng Sun4,
  • Senthilkumar S. Karuppagounder5,6,7,
  • Shuli Xia2,5,
  • Valina L Dawson5,6,7,8,9,
  • Ted M Dawson5,6,7,8,10,
  • John Laterra2,5,8,11,
  • Jianmin Zhang1,
  • Mingyao Ying2,5
  • 1Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology;
  • 2Hugo W. Moser Research Institute at Kennedy Krieger;
  • 3Department of Physiology and Biophysics, Howard University;
  • 4College of Life Sciences, Northwest University;
  • 5Department of Neurology, Johns Hopkins University School of Medicine;
  • 6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine;
  • 7Adrienne Helis Malvin Medical Research Foundation;
  • 8Department of Neuroscience, Johns Hopkins University School of Medicine;
  • 9Department of Physiology, Johns Hopkins University School of Medicine;
  • 10Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine;
  • 11Department of Oncology, Johns Hopkins University School of Medicine
  • Neurodegeneration Method Development Community
    Tech. support email: ndcn-help@chanzuckerberg.com
Anita Broellochs
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External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
Protocol Citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. mRNA Synthesis and Transfection. Protocol exchange https://protocols.io/view/mrna-synthesis-and-transfection-9u5h6y6
Manuscript citation:
Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Xue Y, Zhan X, Sun S, Karuppagounder SS, Xia S, Dawson VL, Dawson TM, Laterra J, Zhang J, Ying M. Stem Cells Transl Med. 2019 Feb;8(2):112-123. doi: 10.1002/sctm.18-0036. Epub 2018 Nov 1. PMID: 30387318
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
These protocols were published in: Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
Created: November 27, 2019
Last Modified: September 16, 2020
Protocol Integer ID: 30333
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, HiScribe T7 ARCA
Abstract
This protocol explains the mRNA synthesis and transfection of lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Guidelines
Please follow the National Institutes of Health guidelines.
Materials
MATERIALS
ReagentHiScribe T7 ARCA mRNA Kit - 20 rxnsNew England BiolabsCatalog #E2065S
ReagentMEGAclear™ Transcription Clean-Up KitThermo FisherCatalog #AM1908
ReagentLipofectamine™ MessengerMAX™ Transfection ReagentThermo FisherCatalog #LMRNA001
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
Before start
Obtain approval to work with human stem cells from an appropriate Institutional Review Board.
mRNA Synthesis
mRNA Synthesis
Clone coding sequence of Atoh1 into a vector containing the T7 promoter and poly(A) tail for in vitro transcription.
Clone coding sequence of Ngn2 into a vector containing the T7 promoter and poly(A) tail for in vitro transcription.
Linearize Atoh1 and Ngn2 vectors.
Digestion
Subject linearized Atoh1 and Ngn2 vectors to mRNA synthesis using the HiScribe T7 ARCA mRNA Kit.
Purify mRNAs using the MEGAclear Transcription Clean‐Up Kit.
Transfect mRNA
Transfect mRNA
Transfect using a cationic lipid (e.g. Stem-In).
For each well of a 12-well plate, incubate Amount0.25 µg mRNA with Amount1.5 µL lipid in Amount50 µL PBS for Duration00:15:00 .


Incubation
Add DNA:lipid complexes to cells.