RT-PCR
The olfactory epithelia were dissected from three adult female C57BL/6 mice, including tissues attached to the skull and septum. RNA was isolated using the Qiagen RNeasy midi kit (Qiagen, Valencia, CA, USA), including a DNase treatment step. First-strand cDNA was produced from 2.5 μg of RNA in a volume of 50 μl using random hexamers and Invitrogen's Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations. One-twenty-fifth of the resulting cDNA was used as template in subsequent PCR reactions. PCR amplification biased towards class I olfactory receptors was performed using degenerate primers P26 [17] and classI_R1 (5'-GGRTTIADIRYIGGNGG-3') with an annealing temperature of 44°C. The product was cloned (TA cloning kit, Invitrogen), and individual clones were sequenced. Specific PCR primers used to confirm expression of individual olfactory receptor genes are given in Additional data file 1. Each PCR product was sequenced to confirm that the expected gene and no others had been amplified. Control reactions on a template made by omitting reverse transcriptase gave no product, indicating that the RNA preparation was uncontaminated by genomic DNA.
Relative transcript levels were estimated using real-time PCR according to Applied Biosystems' protocols, with magnesium concentration, primer pair and fluorescent probe given in Additional data file 2. The increase in fluorescence during thermocycling was measured on an ABI PRISM 7900HT. Standard curves were constructed for each primer pair using triplicate samples of mouse genomic DNA of nine known concentrations (range 0.01-100 ng, or about 3-30,000 copies of the haploid genome). Relative expression level of each gene was determined by comparing the mean Ct (cycle where fluorescence reaches an arbitrary threshold value) obtained with six replicate samples of reverse-transcribed RNA to the standard curve for the corresponding primers. Relative RNA levels of a housekeeping gene, ribosomal S16, were measured as previously described [52]. Control reactions on template prepared by omitting reverse transcriptase amplified at a relative level of 0.03 ± 0.01 ng or less in each case. Expression measurements of the seven genes were normalized for each mouse so that S16 levels were equal to 1 (arbitrary units).