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Identification of putative odorant receptors from Epiphyas postvittana 94 4.2.13 qRT-PCR primer test Each set of primers were used with the cDNA samples from the three different tissues in a gradient cycler to optimise the PCR conditions and obtain the optimal annealing temperature for each. The standard PCR reaction components were as follows: 1x PCR buffer including 1.5 mM magnesium, 0.2 mM of each dNTP, 2 units of Taq DNA polymerase, 0.5 µM of each primer, 30 ng cDNA template and water added to a reaction volume of 20 µL. The PCR cycling conditions were as follows: 95 o C for 2 minutes, followed by 40 cycles at 94 o C for 15 seconds, gradient from 50 to 60 o C for 20 seconds, 72 o C for 30 seconds and a final elongation at 72 o C for 10 minutes. The gradient tested was from 50 o C in column 1 through to 60 o C in column 12. PCR products were visualised on 1% agarose gel with 1x SYBR safe DNA gel stain (Invitrogen) and visualised on the ImageQuant 300 system (GE Healthcare Life Sciences). 4.2.14 Quantitative Real-Time PCR Quantitative real-time PCR were performed in 384 well plates on either Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems) or on the Roche LightCycler 480 instrument (Roche, Germany), with SYBR green as the fluorescent reporter. Each primer template combination was set up in triplicate and a no template water control was included for each primer pair, (two pools of each template giving a total of six different templates). The housekeeping genes Ef1α and α-tubulin (Turner et al., 2006) were used due to their consistent expression in different E. postvittana tissues. For qRT-PCR performed on Applied Biosystems 7900HT Fast Real-Time PCR System, the plate and instrument set up were designed using the SDS 2.1 software (Applied Biosystems). The assay type performed was absolute quantification followed by a dissociation curve analysis at the end of the run to check for single PCR products. The reaction conditions were same as that for primer tests except for the addition of 0.2 µL of 1/1000 SYBR green dye (Molecular Probes). The PCR cycling conditions were 95 o C for 2 minutes, followed by 40 cycles at 94 o C for 15 seconds, 55 o C for 20 seconds, 72 o C for 30 seconds followed by the generation of a dissociation
Identification of putative odorant receptors from Epiphyas postvittana 95 curve with the following thermal profile; 95°C for 15 seconds, 60°C for 15 seconds, 95°C for 15 seconds, with a ramp rate of 2%. The result files obtained from the run were loaded into the SDS 2.1 software and the amplification and dissociation curves were checked for consistency between the triplicates. Wells that had more than one peak in the dissociation curve, hence more than one PCR product were omitted from further analysis. Primer PCR efficiencies were calculated using the LinRegPCR (Ramakers et al., 2003) program and the Cycle Threshold (Ct) values for all the samples were converted to quantity of product formed in each sample by the following equation: Quantity = (PCR efficiency) (minimum quantity for a particular treatment – current Ct value) The mean quantities of the housekeeping genes for each tissue type were used with the software geNorm (Vandesompele et al., 2002) for calculating the normalisation factor for the individual tissue types. These normalisation factors were then used for calculating the relative expression of the samples. For qRT-PCR performed on a Roche LightCycler 480 instrument, the reaction mixture consisted of 5 µL 2x FastStart SYBR Green Master (Roche, Germany), 0.5 µM of each primer and 30 ng of cDNA template, in a total reaction volume of 10 µL made up with nuclease-free water. The thermal profile of the run was as follows: 95 o C for 5 minutes at ramp rate ( o C/s) of 4.8, then 40 cycles at 95 o C for 15 seconds at ramp rate of 4.8, 55 o C for 30 seconds at ramp rate of 2.5 and 72 o C for 20 seconds at ramp rate of 4.8. A melt curve was then generated at the end of the amplification to verify single products with the following profile: 95 o C for 5 seconds at a ramp rate of 4.8, 65 o C for 1 minute at ramp rate of 2.5 followed by continuous acquisition at 97 o C at a ramp rate of 0.11. Analysis of the raw data was carried out with the LightCycler 480 software version 1.5 (Roche). PCR efficiencies of the primers were calculated using the exported amplification data at every cycle using the program LinRegPCR (Ramakers et al., 2003). The relative quantification of the test genes was calculated based on relative expression of target versus housekeeping genes, as stated in Pfaffl (2001).
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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General Introduction 31 BmOr2 HvOr2
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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Page 59 and 60: Functional characterisation of Epip
Page 73 and 74: 3.1 Introduction 3 The roles of Epi
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Page 97 and 98: Identification of putative odorant
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Page 145 and 146: Concluding Discussion 129 redundant
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Page 149 and 150: Concluding Discussion 133 sequencin
Page 151 and 152: Appendix B 135 Buffered TB (Sambroo
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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