PMID- 32161782
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20200928
IS  - 2396-8923 (Electronic)
IS  - 2396-8923 (Linking)
VI  - 1
IP  - 1
DP  - 2016 Mar
TI  - Identification of stable reference genes for lipopolysaccharide-stimulated 
      macrophage gene expression studies.
PG  - bpw005
LID - 10.1093/biomethods/bpw005 [doi]
LID - bpw005
AB  - Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated 
      macrophages to model immune signaling are widely used for elucidating the 
      mechanisms of inflammation-related disease. When expression levels of target 
      genes are quantified using Real-Time quantitative Reverse Transcription 
      Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference 
      genes, which should have stable expression. Judicious selection of reference 
      genes is, therefore, critical to interpretation of qRT-PCR results. Ideal 
      reference genes must be identified for each experimental system and demonstrated 
      to remain constant under the experimental conditions. In this study, we evaluated 
      the stability of eight common reference genes: Beta-2-microglobulin (B2M), 
      Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase 
      (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, 
      TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. 
      Expression stability of each gene was tested under different conditions of LPS 
      stimulation and compared to untreated controls. Reference gene stabilities were 
      analyzed using C(t) value comparison, NormFinder, and geNorm. We found that UBC, 
      closely followed by B2M, is the most stable gene, while the commonly used 
      reference gene GAPDH is the least stable. Thus, for improved accuracy in 
      evaluating gene expression levels, we propose the use of UBC to normalize PCR 
      data from LPS-stimulated macrophages.
CI  - (c) The Author 2016. Published by Oxford University Press. All rights reserved.
FAU - Kalagara, Roshini
AU  - Kalagara R
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
FAU - Gao, Weimin
AU  - Gao W
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
FAU - Glenn, Honor L
AU  - Glenn HL
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
FAU - Ziegler, Colleen
AU  - Ziegler C
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
FAU - Belmont, Laura
AU  - Belmont L
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
FAU - Meldrum, Deirdre R
AU  - Meldrum DR
AD  - Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona 
      State University, Tempe, AZ 85287, USA.
LA  - eng
PT  - Journal Article
DEP - 20161227
PL  - England
TA  - Biol Methods Protoc
JT  - Biology methods & protocols
JID - 101693064
PMC - PMC6994071
OTO - NOTNLM
OT  - J774A.1
OT  - PCR
OT  - housekeeping genes
OT  - inflammation
OT  - murine
OT  - standardization
EDAT- 2016/12/27 00:00
MHDA- 2016/12/27 00:01
PMCR- 2016/12/27
CRDT- 2020/03/13 06:00
PHST- 2016/05/25 00:00 [received]
PHST- 2016/10/12 00:00 [revised]
PHST- 2016/10/13 00:00 [accepted]
PHST- 2020/03/13 06:00 [entrez]
PHST- 2016/12/27 00:00 [pubmed]
PHST- 2016/12/27 00:01 [medline]
PHST- 2016/12/27 00:00 [pmc-release]
AID - bpw005 [pii]
AID - 10.1093/biomethods/bpw005 [doi]
PST - epublish
SO  - Biol Methods Protoc. 2016 Dec 27;1(1):bpw005. doi: 10.1093/biomethods/bpw005. 
      eCollection 2016 Mar.