PMID- 28974200
OWN - NLM
STAT- MEDLINE
DCOM- 20180711
LR  - 20191210
IS  - 1471-2172 (Electronic)
IS  - 1471-2172 (Linking)
VI  - 18
IP  - 1
DP  - 2017 Oct 3
TI  - Selection of reliable reference genes for the normalisation of gene expression 
      levels following time course LPS stimulation of murine bone marrow derived 
      macrophages.
PG  - 43
LID - 10.1186/s12865-017-0223-y [doi]
LID - 43
AB  - BACKGROUND: Macrophages are key players in the initiation, perpetuation and 
      regulation of both innate and adaptive immune responses. They largely perform 
      these roles through modulation of the expression of genes, especially those 
      encoding cytokines. Murine bone marrow derived macrophages (BMDMs) are commonly 
      used as a model macrophage population for the study of immune responses to 
      pro-inflammatory stimuli, notably lipopolysaccharide (LPS), which may be 
      pertinent to the human situation. Evaluation of the temporal responses of LPS 
      stimulated macrophages is widely conducted via the measurement of gene expression 
      levels by RT-qPCR. While providing a robust and sensitive measure of gene 
      expression levels, RT-qPCR relies on the normalisation of gene expression data to 
      a stably expressed reference gene. Generally, a normalisation gene(s) is selected 
      from a list of "traditional" reference genes without validation of expression 
      stability under the specific experimental conditions of the study. In the absence 
      of such validation, and given that many studies use only a single reference gene, 
      the reliability of data is questionable. RESULTS: The stability of expression 
      levels of eight commonly used reference genes was assessed during the peak (6 h) 
      and resolution (24 h) phases of the BMDM response to LPS. Further, this study 
      identified two additional genes, which have not previously been described as 
      reference genes, and the stability of their expression levels during the same 
      phases of the inflammatory response were validated. Importantly, this study 
      demonstrates that certain "traditional" reference genes are in fact regulated by 
      LPS exposure, and, therefore, are not reliable candidates as their inclusion may 
      compromise the accuracy of data interpretation. Testament to this, this study 
      shows that the normalisation of gene expression data using an unstable reference 
      gene greatly affects the experimental data obtained, and, therefore, the ultimate 
      biological conclusions drawn. CONCLUSION: This study reaffirms the importance of 
      validating reference gene stability for individual experimental conditions. Given 
      that gene expression levels in LPS stimulated macrophages is routinely used to 
      infer biological phenomena that are of relevance to human conditions, 
      verification of reference gene expression stability is crucial.
FAU - Tanaka, Akane
AU  - Tanaka A
AD  - The School of Life Sciences, University of Technology Sydney, Ultimo, NSW, 
      Australia.
FAU - To, Joyce
AU  - To J
AD  - The School of Life Sciences, University of Technology Sydney, Ultimo, NSW, 
      Australia.
FAU - O'Brien, Bronwyn
AU  - O'Brien B
AD  - The School of Life Sciences, University of Technology Sydney, Ultimo, NSW, 
      Australia.
AD  - The Centre for Health Technologies, University of Technology Sydney, Ultimo, NSW, 
      Australia.
FAU - Donnelly, Sheila
AU  - Donnelly S
AUID- ORCID: 0000-0003-2005-3698
AD  - The School of Life Sciences, University of Technology Sydney, Ultimo, NSW, 
      Australia.
FAU - Lund, Maria
AU  - Lund M
AD  - The School of Life Sciences, University of Technology Sydney, Ultimo, NSW, 
      Australia. Maria.Lund@uts.edu.au.
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Validation Study
DEP - 20171003
PL  - England
TA  - BMC Immunol
JT  - BMC immunology
JID - 100966980
RN  - 0 (Adjuvants, Immunologic)
RN  - 0 (Lipopolysaccharides)
SB  - IM
MH  - Adjuvants, Immunologic/pharmacology
MH  - Animals
MH  - Cells, Cultured
MH  - Gene Expression/*drug effects/genetics/immunology
MH  - Gene Expression Profiling
MH  - Gene Expression Regulation/*drug effects
MH  - Genes, Essential/genetics/immunology
MH  - Inflammation/chemically induced/*genetics
MH  - Lipopolysaccharides/*pharmacology
MH  - Macrophage Activation/drug effects
MH  - Macrophages/*drug effects
MH  - Mice
MH  - Microarray Analysis
MH  - Reproducibility of Results
MH  - Time Factors
PMC - PMC5627409
OTO - NOTNLM
OT  - Lipopolysaccharide
OT  - Macrophage
OT  - Pro-inflammatory responses
OT  - RT-qPCR
OT  - Reference gene
COIS- ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All experiments were performed in 
      accordance with UTS Animal Care and Ethics Committee guidelines and the study was 
      specifically approved under protocol 2012-080. CONSENT FOR PUBLICATION: Not 
      applicable. COMPETING INTERESTS: The authors declare that they have no competing 
      interests. PUBLISHER'S NOTE: Springer Nature remains neutral with regard to 
      jurisdictional claims in published maps and institutional affiliations.
EDAT- 2017/10/05 06:00
MHDA- 2018/07/12 06:00
PMCR- 2017/10/03
CRDT- 2017/10/05 06:00
PHST- 2017/01/10 00:00 [received]
PHST- 2017/08/01 00:00 [accepted]
PHST- 2017/10/05 06:00 [entrez]
PHST- 2017/10/05 06:00 [pubmed]
PHST- 2018/07/12 06:00 [medline]
PHST- 2017/10/03 00:00 [pmc-release]
AID - 10.1186/s12865-017-0223-y [pii]
AID - 223 [pii]
AID - 10.1186/s12865-017-0223-y [doi]
PST - epublish
SO  - BMC Immunol. 2017 Oct 3;18(1):43. doi: 10.1186/s12865-017-0223-y.