Sep 16, 2020

Public workspaceQuantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR)

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. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR). protocols.io https://protocols.io/view/quantitative-real-time-polymerase-chain-reaction-q-9u8h6zw
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: 30336
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, qRT-PCR
Abstract
This protocol explains Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) of lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Guidelines
Primers for qRT-PCR
Primer Sequence (5'-3')
NEUROD1 AGACACTCGTCTGTCCAGCTT; GCCCCAGGGTTATGAGACTA
FOXA2 GGAACACCACTACGCCTTCAAC; AGTGCATCACCTGTTCGTAGGC
NURR1 AAACTGCCCAGTGGACAAGCGT; GCTCTTCGGTTTCGAGGGCAAA
LMX1A CATCGAGCAGAGTGTCTACAGC; TGTCGTCGCTATCCAGGTCATG
TH GCTGGACAAGTGTCATCACCTG; CCTGTACTGGAAGGCGATCTCA
IFNA ACCCACAGCCTGGATAACAG; ACTGGTTGCCATCAAACTCC
IFNB CATTACCTGAAGGCCAAGGA; CAGCATCTGCTGGTTGAAGA
IFIT1 AAAAGCCCACATTTGAGGTG; GAAATTCCTGAAACCGACCA
OAS1 CGATCCCAGGAGGTATCAGA; TCCAGTCCTCTTCTGCCTGT
PKR TCGCTGGTATCACTCGTCTG; GATTCTGAAGACCGCCAGAG
RIGI GTTGTCCCCATGCTGTTCTT; GCAAGTCTTACATGGCAGCA
18s ACCCGTTGAACCCCATTCGTGA; GCCTCACTAAACCATCCAATCGG
Materials
MATERIALS
ReagentSYBR GreenLife Technologies
ReagentRNeasy Mini KitQiagenCatalog #74104
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.
RNA Extraction
RNA Extraction
Extract total RNA using a RNeasy Mini Kit.
qRT-PCR
qRT-PCR
Reverse transcribe using murine leukemia virus reverse transcriptase and oligo(dT) primers.
Set up qRT-PCR using SYBR green PCR Master Mix in the IQ5 RT‐PCR detection system. Primer sequences are listed in "Guidelines".
Normalize relative expression to 18S rRNA.