Chapter 3: Determination of in planta gene expression by qRT-PCR
2. Establishment of in planta qRT-PCR and selection of primers
qRT-PCR is a useful tool for determination of relative gene expression. However, in planta determination of pathogen V. longisporum gene expression during infection in host plant B. napus had to establish because of diversity of biological organisms and the special infection and colonisation form of V. longisporum, even qRT-PCR is suitable for pathogen biomass in the host in very low level (17). We have improved two procedures for identification of relative gene expression in planta (Figure 2) including “hot phenol” total RNA extraction protocol. one procedure used isolated mRNA for cDNA synthesis, the another used directly total RNA, both cDNAs were purified and concentrated in about 100 ng µl–1 as template of qRT-PCR.
The specificity, amplification efficiency of all used primers for reference as well as putative pathogenicity related genes were validated respectively. They had no crossing-effect with host plant, the amplification efficiency arriving more than 90% and kept less difference between reference and target fragments. It is clear, not a single universal reference gene has been found, it exists yet at all (18). However, for this study ribosomal peptide S17, β-tubulin, GADPH, and histone H3 were selected as stably expressed both in vitro and in planta systems, respectively.
Figure 4a : In planta relative gene expression analasis of selected V. longisporum target
genes Vl_6.2 (TDF6.2), Vl_24.1 (TDF24.1), Vl_12.2(TDF12.2) and Vl_34.2(TDF34.2) in time course after infection. The with total RNA transcripted cDNAs were used as templates for qRT-PCR.Transcript levels of selected Verticillium-genes were measured and normalised using three reference genes, GADPH, ribosomal peptide S17 and histone H3.
Ratios of relative expression levels between Verticillium in vitro as calibrator and in planta as target were calculated as the ΔΔCt values of three biological replicas at each time point.
Error bars indicated the standard deviation of calculated ΔΔCt values. Significance level was set to p values < 0.05 and was calculated for three reference genes, respectively. one asterisk indicates significant change in the expression levels regarding to one of the reference genes.
Figure 4b : In planta relative gene expression analasis of selected V. longisporum target genes Vl_12.1 (TDF12.1), Vl_2.1 (TDF2.1), Vl_30.2(TDF30.2), Vl_11.1(TDF11.1), Vl_PKS1 and Vl_NEP1 in time course after infection. The with total RNA transcripted cDNAs were used as templates for qRT-PCR. Transcript levels of selected Verticillium-genes were measured and normalised using three reference Verticillium-genes, GADPH, ribosomal peptide S17 and histone H3. Ratios of relative expression levels between Verticillium in
vitro as calibrator and in planta as target were calculated as the ΔΔCt values of three biological replicas at each time point. Error bars indicated the standard deviation of calculated ΔΔCt values. Significance level was set to p values < 0.05 and was calculated for three reference genes, respectively.
Figure 5: In planta relative gene expression analysis of selected V. longisporum target
genes Vl_6.2 (TDF6.2), Vl_24.1 (TDF24.1), Vl_12.2 (TDF12.2), Vl_34.2 (TDF34.2), Vl_12.1 (TDF12.1), Vl_2.1 (TDF2.1), Vl_10.2 (TDF10.2), Vl_11.1 (TDF11.1), Vl_PKS1 and Vl_NEP1 in time course after infection. Transcript levels of selected Verticillium-genes were measured and normalised using two reference genes, β-tubulin and ribosomal peptide S17. Ratios of relative expression levels between Verticillium in vitro as calibrator and in planta as target were calculated as the ΔΔCt values of three biological replicas at each time point. Error bars indicated the standard deviation of calculated ΔΔCt values.
Significance level was set to p values < 0.05 and was calculated for two reference genes, respectively. one asterisk indicates significant change in the expression levels regarding to one of the reference genes. The with purified mRNA transcripted cDNAs were used as templates for qRT-PCR. (modified from Weiberg, Xu et al. 2008).
References
1. Bachem, C. W., van der Hoeven, R. S., Bruijn, S. M., Vreugdenhil, D., Zabeau, M. &
Visser, R. G. (1996). Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9: 745-753.
2. Higuchi, R., Fockler, C., Dollinger, G. & Watson, R. (1993). Kinetic PCR: Real time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030.
3. VanGuilder H. D., Vrana, K. E. & Freeman, W. M. (2008). Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques. 44: 619-626.
4. Wang, T. & Brown, M. J. (1999). mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal.
Biochem. 269: 198-201.
5. Wong, M.L. & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation.
BioTechniques. 39: 1-11.
6. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. 29: 2002-2007.
7. Pfaffl, M. W., Horgan, G. W. & Dempfle, L. (2002). Relative expression software tool (REST(C)) for group-wise comparison and statistical analysis of relative expression
results in real-time PCR. Nucl. Acids Res. 30: e36.
8. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods, 25: 402 – 408.
9. Cook, P., Fu, C., Hickey, M., Han, E. S. & Miller, K. S. (2004). SAS programs for real-time RT-PCR having multiple independent samples. Biotechniques. 37: 990-995.
10.Zeise, K. & von Tiedemann, A. (2001). Morphological and Physiological Differentiation among Vegetative Compatibility Groups of Verticillium dahliae in Relation to V. longisporum. J. Phytopathol. 149: 469-475.
11. Zeise, K. & von Tiedemann, A. (2002). Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and Verticillium longisporum. J. Phytopathol.
150:557-563.
12.Williams P. H. & Hill C. B. (1986). Rapid-Cycling Populations of Brassica. Science 232: 1385-1389.
13.Murray, M. G., & Thompson, W. F. (1980). Rapid isolation of high molecular-weight plant DNA. NAR 8: 4321-4325.
14.Eynck, C., Koopmann, B., Grunewalft-Stocker, G., Karlovsky, P. & von Tiedemann A. (2007). Differential interactions of Verticillium longisporum and V. dahliae with Brassica napus detected with molecular and histological techniques. Eur. J. Plant Pathol. 118: 259-274.
15.Yan, H. Z. & Liou, R. F. (2006). Selection of internal control genes for real-time quantitative RT-PCR assays in the oomycete plant pathogen Phytophthora parasitica. Fungal Genetics and Biology. 43 (6): 430-438.
16.Yamamoto, I., Numao, M., Sakaguchi, Y., Tsushima, N. & Tanaka, M. (2007).
Molecular characterization of sequence and expression of chicken GPR39. Gen Comp Endocrinol. 151(1):128-34.
17.Goerke, C., Bayer, M. G. & Wolz, C. (2001). Quantification of bacterial transcripts during infection using competitive reverse transcription-PCR (RT-PCR) and
LightCycler RT-PCR. Clin. Disgn. Lab. Immun. 8: 279-282.
18.Andersen, C. L., Jensen, J. L. & Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcrition-pcr data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research. 64: 5245-5250.
19.Chandler, D. P., Wagnon, C. A. & Bolton, H. Jr. (1998) Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR. Appl. Environ. Microbiol. 64: 669.
20.Engel, H., Küppers, C. & Löffert, D. (2003) Highly efficient multiplex PCR using novel reaction chemistry. QIAGEN News 2003, 41.
21.Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H., & Vogt, P.H. (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504.
22.Higuchi, R., Dollinger, G., Walsh, P. S., & Griffith, R. (1992). Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413–417.
23.Logan J, Edwards K, & Saunders, N. (2009). Real-Time PCR: Current Technology and Applications. Caister Academic Press.ISBN 978-1-904455-39-4.
24.Mackay, I. M. (2007). Real-Time PCR in Microbiology. Caister Academic Press.
ISBN 978-1-904455-18-9
25.Multiplex PCR that simply works ― the new QIAGEN Multiplex PCR Kit. QIAGEN News 2002, No. 5, 1.
26.Rita S. Cha & William G. Thilly. (1993). Specificity, Efficiency, and Fidelity of PCR.
Genome Res. 3: 18-29. Cold Spring Harbor Laboratory Press.
27.Sambrook, J, Fritsch, E, & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press.
28.Wawrik, B., Paul, J. H., Tabita, F. R. (2002). Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. Appl. Environ. Microbiol. 68: 3771-3779.