Quantitative RT-PCR using the TaqMan® Gene Expression Assays on  StepOnePlus™ Real-Time PCR System

Lisa-Maria Rosenthal; Giang Tong; Katharina Schmitt

Jun 04, 2019

Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System

PLOS One

DOI

dx.doi.org/10.17504/protocols.io.2v8ge9w

Lisa-Maria Rosenthal¹,
Giang Tong¹,
Katharina Schmitt¹

¹Deutsches Herzzentrum Berlin

Lisa-Maria Rosenthal

Deutsches Herzzentrum Berlin

DOI: dx.doi.org/10.17504/protocols.io.2v8ge9w

External link: https://doi.org/10.1371/journal.pone.0226005

Protocol Citation: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt 2019. Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System. protocols.io https://dx.doi.org/10.17504/protocols.io.2v8ge9w

Manuscript citation:

Rosenthal L,  Leithner C,  Tong G,  Streitberger KJ,  Krech J,  Storm C,  Schmitt KRL (2019) RBM3 and CIRP expressions in targeted temperature management treated cardiac arrest patients—A prospective single center study. PLoS ONE  14(12): e0226005. doi: 10.1371/journal.pone.0226005

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

We use this protocol and it's working

Created: May 15, 2019

Last Modified: June 04, 2019

Protocol Integer ID: 23200

Abstract

Quantitative RT-PCR using the TaqMan® Gene Expression Assays on 
StepOnePlus™ Real-Time PCR System

Materials

MATERIALS
ReagentTaqMan® Gene Expression AssayThermo Fisher ScientificCatalog #4331182 
ReagentStepOnePlus™ Real-Time PCR System Thermo Fisher ScientificCatalog #4376600 

 
  Allow the TaqMan® Gene Expression Assay components to thaw on ice

Primers

Primers
GeneGene Assay IDGeneGene Assay ID
RBM3Hs00943160_g1CIRPHs00989762_g1
IL-6Hs00174131_m1MCP-1 (Ccl2)Hs00234140_m1
iNOSHs01075529_m1GAPDHHs02786624_g1

P

  Prepare the TaqMan® master mix on ice.
 
ComponentVolume (µL)
Gene Assay Primers0.5
TaqMan Gene Expression PCR Master Mix5.0
cDNA0.5
Nuclease-free H2O4.0
Total per reaction10.0

  Run the StepOnePlus™ Real-Time PCR System under the following conditions.
 
StepTimes and Temperatures
2. PCR StepInitial StepsPCR (40 Cycles)
AmpErase ® UNG ActivationAmpliTaq Gold® DNA Polymerase ActivationMeltAnneal/ Extend
HOLDHOLDCYCLE
2 min @ 50 °C10 min @ 95 °C15 sec @ 95 °C1 min @ 60 °C

Primers
Gene	Gene Assay ID	Gene	Gene Assay ID
RBM3	Hs00943160_g1	CIRP	Hs00989762_g1
IL-6	Hs00174131_m1	MCP-1 (Ccl2)	Hs00234140_m1
iNOS	Hs01075529_m1	GAPDH	Hs02786624_g1

	Component	Volume (µL)
	Gene Assay Primers	0.5
	TaqMan Gene Expression PCR Master Mix	5.0
	cDNA	0.5
	Nuclease-free H2O	4.0
	Total per reaction	10.0

Step	Times and Temperatures
2. PCR Step	Initial Steps		PCR (40 Cycles)
	AmpErase ® UNG Activation	AmpliTaq Gold® DNA Polymerase Activation	Melt	Anneal/ Extend
	HOLD	HOLD	CYCLE
	2 min @ 50 °C	10 min @ 95 °C	15 sec @ 95 °C	1 min @ 60 °C

Public workspaceQuantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System

Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System