PMID- 20587017
OWN - NLM
STAT- MEDLINE
DCOM- 20101220
LR  - 20211020
IS  - 1471-2199 (Electronic)
IS  - 1471-2199 (Linking)
VI  - 11
DP  - 2010 Jun 29
TI  - Predator-induced defences in Daphnia pulex: selection and evaluation of internal 
      reference genes for gene expression studies with real-time PCR.
PG  - 50
LID - 10.1186/1471-2199-11-50 [doi]
AB  - BACKGROUND: The planktonic microcrustacean Daphnia pulex is among the 
      best-studied animals in ecological, toxicological and evolutionary research. One 
      aspect that has sustained interest in the study system is the ability of D. pulex 
      to develop inducible defence structures when exposed to predators, such as the 
      phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex 
      is accelerating research to identify genes that confer plastic phenotypes that 
      are regularly cued by environmental stimuli. Yet for quantifying gene expression 
      levels, no experimentally validated set of internal control genes exists for the 
      accurate normalization of qRT-PCR data. RESULTS: In this study, we tested six 
      candidate reference genes for normalizing transcription levels of D. pulex genes; 
      alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box 
      binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and 
      CAPON, a protein associated with the neuronal nitric oxide synthase, were 
      selected on the basis of an earlier study and from microarray studies. One 
      additional gene, a matrix metalloproteinase (MMP), was tested to validate its 
      transcriptional response to Chaoborus, which was earlier observed in a microarray 
      study. The transcription profiles of these seven genes were assessed by qRT-PCR 
      from RNA of juvenile D. pulex that showed induced defences in comparison to 
      untreated control animals. We tested the individual suitability of genes for 
      expression normalization using the programs geNorm, NormFinder and BestKeeper. 
      Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. 
      Analyses on the relative expression level using the software REST showed that 
      both classical housekeeping candidate genes (aTub and GAPDH) were significantly 
      downregulated, whereas the MMP gene was shown to be significantly upregulated, as 
      predicted. aTub is a particularly ill suited reference gene because five copies 
      are found in the D. pulex genome sequence. When applying aTub for expression 
      normalization Xbp1 and Tbp are falsely reported as significantly upregulated. 
      CONCLUSIONS: Our results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are 
      suitable reference genes for accurate normalization in qRT-PCR studies using 
      Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the 
      importance of verifying the expression stability of putative reference genes for 
      normalization of expression levels.
FAU - Spanier, Katina I
AU  - Spanier KI
AD  - Department of Animal Ecology, Ruhr-University Bochum, D-44780 Bochum, Germany.
FAU - Leese, Florian
AU  - Leese F
FAU - Mayer, Christoph
AU  - Mayer C
FAU - Colbourne, John K
AU  - Colbourne JK
FAU - Gilbert, Don
AU  - Gilbert D
FAU - Pfrender, Michael E
AU  - Pfrender ME
FAU - Tollrian, Ralph
AU  - Tollrian R
LA  - eng
GR  - 5R24GM078274/GM/NIGMS NIH HHS/United States
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20100629
PL  - England
TA  - BMC Mol Biol
JT  - BMC molecular biology
JID - 100966983
RN  - 0 (Insect Proteins)
RN  - 0 (Tubulin)
RN  - 63231-63-0 (RNA)
RN  - EC 3.4.24.- (Matrix Metalloproteinases)
SB  - IM
MH  - Animals
MH  - *Daphnia/genetics/physiology
MH  - Diptera/physiology
MH  - Female
MH  - *Gene Expression
MH  - Gene Expression Profiling/methods/*standards
MH  - *Gene Expression Regulation
MH  - Genes, Insect
MH  - Insect Proteins/genetics
MH  - Matrix Metalloproteinases/genetics
MH  - *Polymerase Chain Reaction/methods/standards
MH  - Predatory Behavior/*physiology
MH  - RNA/genetics
MH  - Reference Standards
MH  - Tubulin/genetics
PMC - PMC3148505
EDAT- 2010/07/01 06:00
MHDA- 2010/12/21 06:00
PMCR- 2010/06/29
CRDT- 2010/07/01 06:00
PHST- 2009/08/31 00:00 [received]
PHST- 2010/06/29 00:00 [accepted]
PHST- 2010/07/01 06:00 [entrez]
PHST- 2010/07/01 06:00 [pubmed]
PHST- 2010/12/21 06:00 [medline]
PHST- 2010/06/29 00:00 [pmc-release]
AID - 1471-2199-11-50 [pii]
AID - 10.1186/1471-2199-11-50 [doi]
PST - epublish
SO  - BMC Mol Biol. 2010 Jun 29;11:50. doi: 10.1186/1471-2199-11-50.