Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
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Identification <strong>of</strong> putative odorant receptors from Epiphyas postvittana 86<br />
4.2 Materials and Methods<br />
4.2.1 Materials<br />
All the PCR reagents were from Invitrogen unless otherwise stated.<br />
4.2.2 Total RNA Extraction<br />
RNA was extracted from two pools <strong>of</strong> 100 antennae pairs each for male and female E.<br />
postivttana and from two pools <strong>of</strong> moth female body without the head, with three<br />
bodies per pool. TRIzol Reagent (Invitrogen) was used for isolat<strong>in</strong>g RNA follow<strong>in</strong>g<br />
manufacturer‟s <strong>in</strong>structions, and the result<strong>in</strong>g pellet resuspended <strong>in</strong> 20 µL <strong>of</strong> TE<br />
buffer. The concentration <strong>of</strong> the samples was measured on a nanodrop at an optical<br />
density <strong>of</strong> 260-280 (OD260/280) (Thermo Sce<strong>in</strong>tific) and the samples stored at -80°C<br />
until use.<br />
4.2.3 mRNA Isolation<br />
mRNA from male and female E. postvittana antennae was isolated us<strong>in</strong>g the Ambion<br />
MicroPoly(A)Purist Kit (Applied Biosystems) and the quality assessed on an<br />
Agilent 2100 Bioanalyzer, follow<strong>in</strong>g manufacturer‟s <strong>in</strong>structions.<br />
4.2.4 cDNA Synthesis<br />
cDNA was synthesised from 1µg <strong>of</strong> RNA us<strong>in</strong>g either Superscript III reverse<br />
transcript (Invitrogen) or the iscript cDNA synthesis kit (Bio-Rad), follow<strong>in</strong>g<br />
manufacturer‟s <strong>in</strong>structions. A no reverse transcriptase control was <strong>in</strong>cluded for every<br />
sample, and the synthesised cDNA stored at -20°C until required.<br />
4.2.5 Degenerate PCR<br />
To identify conserved regions between E. postvittana OR1 and ORs from three other<br />
<strong>in</strong>sect species belong<strong>in</strong>g to the same phylogenetic clade (M. sexta, B. mori and H.<br />
virescens), multiple sequence alignment <strong>of</strong> the follow<strong>in</strong>g OR prote<strong>in</strong> sequences were<br />
conducted us<strong>in</strong>g clustalX 1.81 (MsOR1, BmOR3, HvOR6, HvOR16, HvOR14,