Accurate quantification of mRNAs and housekeeping gene selection for qRT-PCR

normalization in European beech (Fagus sylvatica L.) during

abiotic and biotic stress

Maren OlbrichMaren.olbrich@gsf.de

Institute for Biochemical Plant Pathology - Molecular Phytodiagnostic

GSF-National Research Center for Environment and Health, Neuherberg, Germany

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Issue

• The most important deciduous tree in Central Europe

• Limited genetic and genomic information is available

• High genomic diverstiy

• Long generation times

• Identification of genes specifically up- or down-regulated in plants upon environmental stimuli

• Represents a key step in understanding of ecotoxicologicalprocesses and the systemic results of abiotic and biotic stress

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Issue

• Generated a microarray with ozone-responsive EST‘s

Olbrich M, Betz G, Gerstner E, Langebartels C, Sandermann H, Ernst D (2005) Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.). Plant Biol 7:670-676

• Ozone is an abiotic elicitor of plant defence, well studied in herbaceous plants

• Investigation of transcriptional regulation upon environmental stress, with microarray-technology

Materials, Methods and Results

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Ubiquitin-like protein

α-tubulin

GAPDH2

GAPDH1

Actin

18S rRNA

Reference Genes

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Stress Scenarios

Phytophthora citricolainfection

Picture from tree: Frank Fleischmann

2x ambient CO2

2x ambient O3

Shade leaves

Sun leaves

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

RNA-Isolation and RT-PCR

• RNA isolation from leaves according to Kiefer et al. 2000

• Yield and quality of RNA using Nano Drop system (Kisker, Steinfurt, Germany)

• RT-PCR: oligo dT and Superscript III (Invitrogen, Karlsruhe, Germany)

• Digest with RNAse H and A

Kiefer E, Heller W, Ernst D (2000) A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Mol Biol Rep 18:33-39

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

qRT-PCR

• Primerdesign: Primer Express 2.0® (Applied Biosystems)

• qRT-PCR with Sybr Green PCR kit (ABgene, Hamburg, Germany)

• 7500 Real-Time PCR System, Applied Biosystems (Darmstadt, Germany)

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Statistical analysis

• Analysis of variance, using a linear model

• Specific structure of data (no identical genotyps of beech trees) the data were grouped with respect to trees (technical replicates)

• Linear mixed-effects model, nlme package of R-system

• testing of variance homogeneity (diagnostic plot), normal and indepentent distribution of residuals

R Development Core Team (2005) A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna, Austria

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

PCR efficiency

• Efficiencies of target and reference genes should be approximately equal

• 2-fold dilution series display ΔCt values of 1 between adjacent dilution, relfecting a PCR efficiency of 100 %

• increasing cDNA concentration on the axis result in a graph with a slope -1

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

qPCR effciency

y = -0.929x + 10.01R2 = 0.9968

y = -0.9131x + 22.346R2 = 0.9975

y = -1.0274x + 23.386R2 = 0.9999

y = -1.039x + 24.341R2 = 0.9999

0

5

10

15

20

25

0.0031 0.0063 0.0125 0.025 0.05 0.1

cDNA-concentration in ng

Ct-v

alue

18S rRNA Actin GAPDH1 α-TubulinLi (18S RNA) Li (A ti ) Li (GAPDH1) Li ( T b li )

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Phytophthora and CO2

• Three year old European Beech (Fagus sylvatica L.)

• Climate chamber: 360 and 780 ppm CO2

• Direct inocculation of soil with P. citricola, flooding of the container

• Samples were taken at 14h, 31h, 12d after infection (F. Fleischmann, W. Oßwald, TU München, Ökophysiologie)

Fleischmann F, Schneider D, Matyssek R, Oßwald WF (2002) Investigations on net CO2 assimilation, transpiration and root growth of Fagus sylvatica infested with four different Phytophthora species. Plant Biology 4:144-152.

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Exposure of European beechtrees with 2x ambient O3

Control plants

Greenhouse 2006 - ozone

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Kranzberg Forest – sun and shade leaves

• Sampling of sun and shaded leaves• (P. Fabian, H. Werner, B4: K. H. Häberle, A1: G. Bahnweg, C. Knappe, GSF)

Pretzsch H, Kahn M, Grote R (1998) The mixed spruce-beech forest stands of the "Sonderforschungsbereich" "Growth or Parasite Defence?" in the forest district Kranzberger Forst. Forstwissensch. Zentralblatt 117:241-257

3rd international q-PCR Event, TUM-Freising - Maren Olbrich 28.03.07

Conclusion

• Actin appears to be the best reference gene for European beech under the conditions tested

• Transcriptional level of plant genes is influenced by many factors

• For a single experiment a reference gene must be validated

Thanks to TUM: Kranzberg Forest

Rainer MatyssekKarl-Heinz Häberle

GSF: GreenhouseGünther BahnwegClaudia Knappe

StatisticGerhard Welzl

Dieter Ernst, GSF

Elke Gerstner, GSF

Climate chambersFrank Fleischmann Wolfgang Oßwald