RNA profiles of rat olfactory epithelia: individual and age related variations
© Rimbault et al; licensee BioMed Central Ltd. 2009
Received: 21 August 2009
Accepted: 2 December 2009
Published: 2 December 2009
Mammalian genomes contain a large number (~1000) of olfactory receptor (OR) genes, many of which (20 to 50%) are pseudogenes. OR gene transcription is not restricted to the olfactory epithelium, but is found in numerous tissues. Using microarray hybridization and RTqPCR, we analyzed the mRNA profiles of the olfactory epithelium of male and female Brown Norway rats of different origins and ages (newborn, adult and old).
(1) We observed very little difference between males and females and between rats from two different suppliers. (2) Different OR genes were expressed at varying levels, rather than uniformly across the four endoturbinates. (3) A large proportion of the gene transcripts (2/3 of all probes) were detected in all three age groups. Adult and older rats expressed similar numbers of OR genes, both expressing more OR genes than newborns. (4) Comparisons of whole transcriptomes or transcription profiles of expressed OR genes only showed a clear clustering of the samples as a function of age. (5) Most OR genes were expressed at lower levels at birth than in older animals, but a small number of OR genes were expressed specifically or were overexpressed in newborns.
Not all OR genes are expressed at a detectable level. Pups expressed fewer OR genes than adult rats, and generally at a lower level; however, a small subset of OR genes were more strongly expressed in these newborn rats. The reasons for these differences are not understood. However, the specific expression of some OR genes in newborn olfactory epithelia may be related to the blindness and deafness of pups at birth, when these pups are heavily reliant on olfaction and their mother.
Olfactory receptor (OR) genes were first identified in the rat nasal epithelium by Buck and Axel in 1991 . The receptors they encode play an essential role in olfaction, constituting a key initial element in a cascade of biochemical reactions that lead to odorant perception and recognition. Gene cloning and in silico mining of a number of mammalian genome sequences have identified about 800 OR intact genes and pseudogenes in the human genome [2, 3] and up to 1500 OR genes (including pseudogenes) in the rat genome . Indeed, these genes constitute by far the largest gene family in mammalian genomes.
A substantial percentage of OR genes -- 50% in humans , 24% in mouse , 20.3% in dog and 19.5% in rat  -- are pseudogenes. However, the distinction between pseudogenes and potentially active genes is not strictly defined: for example, a particular gene may exist as either a pseudogene or a potentially active gene, depending upon the population or the individual, as shown for dogs  and for humans .
Only a small number of OR proteins have been deorphanized, i.e. the ligand that they bind to has been identified, and the role of the vast majority of these proteins in olfaction remains undefined. Cloning experiments and microarray analyses have added an additional layer of complexity by showing that at least some pseudogenes can be transcribed [9, 10] and that the transcription of OR genes is not necessarily restricted to the nasal mucosa, but is also detected in several other tissues, including testis  and kidney .
ORs are expressed on the surface of the cilia of olfactory sensory neurons (OSNs) lining the neuroepithelium in the nasal cavity, the site of odorant inhalation . Each OSN expresses one OR from a single allele [14–18]; their axons extend to the olfactory bulb, where the axons of all OSN expressing the same OR converge on a single glomerulus [19–21]. The mechanism by which axons of OSNs expressing the same OR, but dispersed along the olfactory epithelium, converge on the same glomerulus is not totally understood. However, ORs that are not restricted to OSN cilia, but which are also present at the tip of the axons [22, 23] contribute, together with other proteins, to the coalescence of the axons [21, 24–26].
Studies based on cDNA library analysis and microarray hybridization have shown that only a subset of the mouse OR gene repertoire is expressed at detectable levels in the olfactory epithelium [9, 27]. In humans, the pattern of transcription differs slightly between individuals . The importance of such differences is not known. In particular, it is unclear whether these differences reflect individual differences in sensory function, possibly related to some form of anosmia or hyperosmia, or whether they reflect environmental differences. In this study, we carried out hybridization on whole rat genome microarrays, to analyze the transcriptome of the olfactory epithelium of adult Brown Norway rats of different origins and sex. The transcriptomes of these rats were then compared with those of newborn and aged rats, to investigate changes associated with aging.
Results and Discussion
I - The olfactory epithelium transcriptome of adult Brown Norway rats
For probes that did not give either a positive or negative signal (above or below background levels, respectively) for all samples, we compared the signal status of each probe (positive or negative) in the right and left samples from each rat. We observed two different scenarios for the 14 samples (corresponding to seven rats from which we obtained the right and left samples): identical responses in the left and right samples, and different responses for the left and right samples. For 668 probes, we observed a 12+/2- distribution (12 samples showing positive signal and two showing negative results); for 83 of these 668 probes, the two negative results were obtained for left and right samples from the same rat. Similarly, a 12-/2+ distribution of negative and positive signal was observed for 383 probes; 105 of these probes yielded two positive results for the left and right samples from a single rat. Statistical analysis (binomial test, p value < 10-4) of the data clearly demonstrated that both negative and positive results were more frequently paired (right and left samples from the same animal giving identical results) than would be expected if the distribution were random. Probes giving different results for different samples presumably corresponded to two groups of transcripts: one corresponding to a group of genes poorly expressed at levels close to the detection limit of the method used, generating a random distribution of negative and positive results; and a second group corresponding to genes giving the same results for the right and left samples of an individual (either both positive or both negative), and thus clearly expressed by some animals and not expressed by others.
As discussed in more detail below, the set of probes corresponding to weakly expressed or not expressed genes in these experiments depends on the sensitivity of the detection method used. Nevertheless, the proportion of OR genes considered to be expressed at detectable levels in this study is consistent with previous suggestions that up to 76% of OR genes are expressed at a detectable level in the human olfactory epithelium .
• Statistical analysis (t-test)
We performed a statistical analysis (t-test, p value < 0.01) to identify genes that were differentially expressed between animals from two different suppliers and between males and females.
Genes differentially expressed in the olfactory epithelia of rats from different suppliers
Fold difference Charles River/Janvier
period homolog 3
hippocampus abundant transcript-like 1
period homolog 3
D site of albumin promoter (albumin D-box) binding protein
RAB3 GTPase activating protein subunit 2
natriuretic peptide precursor C
chemokine (C-X-C motif) ligand 1
Genes differentially expressed in the olfactory epithelia of male and female rats
Fold difference Female/Male
ubiquitously transcribed tetratricopeptide repeat, X chromosome
coiled-coil domain containing 39
eukaryotic translation initiation factor 2, subunit 3, structural gene X-linked
eukaryotic translation initiation factor 2, subunit 3, structural gene X-linked
We performed hierarchical clustering and principal component analysis for the whole set of expressed gene transcripts and with OR genes only (data not shown). The absence of clear clustering using either of these two approaches, together with only a very small number of genes identified in the t-test analysis, clearly demonstrates that all animals expressed essentially the same genes to similar levels, regardless of their origin and sex.
• Real-time reverse-transcription PCR analysis (RTqPCR)
II - Changes in mRNA profile with aging
Comparison of the lists of gene transcripts and OR gene transcripts only showed that a vast majority of them are common to all three age groups. However, there are some notable differences between newborn and the two other groups: 332 OR genes were expressed by adults and older animals but not by newborn rats and nine OR genes were expressed by all newborn rats only (see Venn diagrams in additional file 2). Considering each litter separately, between 15 and 23 OR genes (depending upon the litter) were identified as expressed in newborns only (additional file 3).
OR genes upregulated in newborns
L1 vs Adult
L1 vs Old
L2 vs Adult
L2 vs Old
L3 vs Adult
L3 vs Old
L4 vs Adult
L4 vs Old
Adult vs Old
These findings -- the results of the statistical analysis (t-test; Table 3) identifying eight OR genes more strongly expressed in newborn rats than in adults, the results of RTqPCR, showing five genes overexpressed in newborn rats (Figure 10) and those presented in Figure 6 (n = 3) -- suggest that a small set of OR genes are more strongly expressed in newborn animals, with the vast majority expressed at a lower level in these animals.
We report patterns of gene expression in the olfactory epithelium of adult Brown Norway rats. We found that 2/3 of the probes (i.e. 26,701 probes spotted on the arrays) gave a hybridization signal identifying genes expressed at a detectable level. The corresponding genes included 732 OR genes (65% of the total OR repertoire). We also showed by clustering analysis of the transcripts expressed in common that the pattern of expression depended on the age of the animal rather than on individual characteristics. The number of genes expressed in the olfactory epithelium, whatever their coding function, was found not to differ significantly from those reported for other tissues  or from the number of OR genes expressed in the mouse or human olfactory epithelium [9, 10, 27]. However, the classification of a gene as expressed or not expressed depends on both the detection threshold and the analytical methods used. Different arrays made with different probes may give slightly different results, as some genes not detected with one brand of microarrays may be detected by another brand due to different hybridization conditions or probe characteristics. Furthermore high-throughput sequencing, depending upon the sequencing depth, can be expected to extend the list of expressed genes . Extending the list of weakly expressed OR genes is also likely to increase the ratio between the most and least strongly expressed genes.
Extending the list of poorly expressed OR genes will raise many questions. What is the minimum level at which an OR gene must be expressed to induce a signal recognized and processed by the brain? Why are some OR genes strongly expressed, whereas others are expressed only very weakly, if at all? Is this a consequence of the environment or does the panel of expressed OR genes represent the minimum required for the recognition of all relevant odorants, including those not yet encountered? In this case, under what circumstances may the transcription of weakly or not expressed OR genes be up-regulated? Answers to these questions might be obtained by subjecting rats to different olfactory environments.
Little difference was found between the olfactory epithelium mRNA profiles of individual Brown Norway adult rats of the same age, sex and origin, allowing a reference transcriptome to be defined. However, the RNA profiles of newborn, adult and old rats showed marked differences: both the lists of genes specifically expressed at each age group and the levels of expression of genes expressed in all three age groups differed between the three groups, allowing a clear clustering of the samples as a function of age. Although 22-month old rats may not be considered very old, it should be noted that the life expectancy of male Brown Norway rats is only around 31 months . The gradual loss of olfactory responses in old age is probably at least in part due to the loss of central brain function [42, 43]. However, the small but measurable changes in the mRNA profiles of the olfactory epithelium observed in this study between adults and old rats may also contribute to this deterioration. The smaller number of OR genes expressed and their lower levels of expression at birth would be consistent with an incomplete development of olfactory function at this age.
The Venn diagrams, t-tests and RTqPCR analyses reported here all indicate that a small number of OR genes (n = 16) were more strongly expressed or expressed exclusively in newborn rats from different litters. This number is likely to be an underestimation considering that five of the 77 OR genes taken at random were overexpressed at birth. The types of ligand they recognize are not known, but given that two- to five-day-old rats are blind and deaf, these OR genes may be important for behavior, mother-pup relationship and/or nipple recognition. These findings are consistent with the recent observation that newborn rats react to odorant exposure .
Brown Norway rats were obtained from Charles River Laboratories (L'Arbresle, France) or Elevage Janvier (Le Genest-Saint-Isle, France). Female rats with their progeny (3 to 5 days old) were purchased from Charles River Laboratories. From their arrival until the time at which they were killed, the rats were kept in the animal house (12:12 h light/dark cycles with free access to food and water) under the rules established by the Board of the Ethical Committee.
Olfactory epithelium dissection
Rats were anesthetized with an injection of 0.3 ml/100 g body weight ketamine hydrochloride (Clorketam 1000 from Vetoquinol). They were then killed by decapitation. Rat skulls were opened through a sagittal section and right and left olfactory epithelia were quickly removed and placed separately in RA1 Buffer from the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany).
Total RNA was isolated with the Nucleospin RNA kit, according to the manufacturer's (Macherey-Nagel, Düren, Germany) instructions, which included an in-column DNase treatment before RNA elution, to ensure the absence of genomic DNA. Recovered RNA was quantified with a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Cambridge, UK), and RNA integrity was assessed with the RNA 6000 Nano LabChip kit, using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto CA, USA). Only RNA samples with an RNA Integrity Number (RIN) greater than 8.8 were used for further analysis (RNA profiling analysis and real time reverse transcription PCR analysis). Application of this strict quality threshold resulted in the elimination of the left sample from one male adult rat from Elevage Janvier.
Target preparation and microarray hybridization
RNA samples were labeled with the Agilent Low RNA Input Fluorescent Linear Amplification kit (p/n 5184-3523), according to the manufacturer's instructions. Briefly, 350 ng of total RNA was used as template for reverse transcription to generate cDNA, which was transcribed with T7-polymerase; cyanine-3 (Cy3)-labeled CTP was used for labeling. Cy3 labeling was monitored with a Nanodrop ND-1000 spectrophotometer and was found to be between 1.2 and 1.9 pmol/μl.
Hybridization was performed with the Agilent Gene Expression Hybridization kit (p/n 5188-5242), used according to the manufacturer's instructions. Briefly, 1650 ng of labeled cRNA from each RNA sample was mixed with Hybridization Buffer and Blocking Agent and subjected to fragmentation (by incubation for 30 min at 60°C in the dark). Hybridizations onto 4 × 44K Whole Rat Genome 60-mer oligonucleotide microarrays (G4131F) (Agilent Technologies, Palo Alto CA, USA) were performed in a rotary oven (65°C, 17 h and 10 rpm) in the dark. Slides were disassembled and washed in Gene Expression Wash Buffers I and II, according to the manufacturer's instructions, and dried with a nitrogen-filled air gun before scanning. Fifteen arrays were used for the experiment analyzing the male/female and rat origin comparisons. Thirty-five arrays were used for the "aging" comparison.
Data acquisition and processing
Microarrays were scanned with a dynamic autofocus microarray scanner (Agilent DNA Microarray Scanner), using Agilent parameters. Feature Extraction software version 9.5 was used to extract and analyze the signals. Array results were analyzed with GeneSpring GX software version 7.3 (Agilent Technologies), via the Enhanced Agilent Feature Extraction Import Preprocessor.
Data were normalized in two steps, using the algorithms supplied with the Feature Extraction software. Data were first transformed to convert any negative value to 0.01; normalization was then performed by a per-chip 50th percentile method, which normalized the data for each chip with respect to the median of the chip concerned, allowing comparison between chips. A second normalization step was applied to the results for each gene across all the arrays in the study ("normalize to median"): the median of all the values obtained for a given gene was calculated and used as the normalization standard for that gene, such that, regardless of absolute differences in the expression of the various genes, all were placed on the same scale for comparison.
The accuracy of microarray results was assessed by comparing the overall gene expression levels for each chip by box plot analysis. Each box plot was centered on zero, with comparable dynamic intensities, demonstrating the technical homogeneity of the experiment overall (data not shown).
The microarray data have been uploaded into the Gene Expression Omnibus (GEO) database (SuperSeries no. GSE15954 and samples nos. GSM400094-GSM400143).
Low-intensity and unreliable results were removed using a "filter on flags" feature, with standardized software algorithms classifying spots as "present," "marginal," or "absent". Spots were considered "present" only if the output was uniform, not saturated and significantly above background; spots that satisfied the main requirements but were outliers relative to the typical values for the other genes were considered "marginal". Filters were set to retain for further analysis only those values classified as "present" or "marginal".
The terms "present" or "marginal" defining the nature of the hybridization signals on each microarray should not be confused with the terms expressed transcripts, weakly expressed transcripts and not expressed transcripts defined by comparing the results obtained with the different samples, as explained in Figure 1.
Content of the 44K Agilent microarrays
There are currently 44,012 probes on each microarrays. By annotation assignments , accession numbers could be assigned to 39,308 of the 39,688 probes for which the manufacturer provided chromosomal location information: rat GenBank accession numbers were assigned for 36,383 of the probes; rat Ensembl transcript identifications (IDs) were assigned for 168 other probes; and non-rat accession numbers for 2,757 probes for which no rat annotations were available. Together, these probes encompass 23,642 unique rat accession numbers and 2,270 unique non-rat accession numbers and represent 16,947 rat Unigene IDs plus 5,941 non-rat Unigene IDs (Unigene build 166). In addition to these probes, there are a number of so-called technical probes engineered by Agilent and used by GeneSpring to ascertain the quality of the data. For additional details, please consult the Agilent website .
Due to uncertainties regarding the names of a number of genes that are probed by many oligonucleotides on the arrays, the term "gene transcripts", used throughout this paper, designates transcripts and genes collectively identified by these probes, except for OR genes that are annotated as such. Although some gene transcripts were probed by more than one oligonucleotide, each OR gene was probed by a single oligonucleotide.
Selection of differentially expressed genes
We performed t-test analysis with GeneSpring software (Benjamini & Hochberg correction for false discovery rate (p value of 0.01)) to select genes that were differentially expressed between groups.
Hierarchical support trees including bootstrap analysis with replacement after 1000 iterations were constructed with TIGR Mev v 4.2 software . Numbers at the nodes (range = 1 to 100) indicate the support for the clustering. The clustering pattern was generated by Pearson Correlation with average linkage clustering.
Analysis of the enrichment of expressed genes with Gene Ontology (GO) categories (i.e. GO terms with a significantly larger number of associated genes than expected for a random distribution) was performed with NIH DAVID [48, 49]. Briefly, the GenBank accession numbers of the genes of interest were uploaded to the DAVID website and analysis was carried out with the Rattus norvegicus gene repertoire as a reference list. GO categories with significantly larger numbers of expressed genes than expected (p value corrected < 0.05) were selected.
Real time reverse transcription PCR analysis (RTqPCR)
RTqPCR was performed for a number of genes, with forward (F) and reverse (R) primers designed with Primer3 software  (additional file 8). Primer specificity was assessed from the monophase dissociation curves. Only pairs presenting similar efficiencies (100 ± 5%) were retained (data not shown). Briefly, the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription and the Power SYBR Green PCR master kit (Applied Biosystems) was used for quantitative PCR, according to Applied Biosystems gene amplification specifications (40 cycles of 15 s at 95°C and 1 min at 60°C). Gene expression was analyzed with the ABI Prism 7900HT sequence detection system, and results were handled with the associated SDS version 2.3 software (Applied Biosystems).
Hprt (hypoxanthine-guanine phosphoribosyltransferase) mRNA levels did not vary significantly between groups or experiments. This gene was therefore used as an internal reference for the comparison of rats of different origins and ages. The relative amounts of gene transcripts were determined by the Ct method . Each PCR was carried out in triplicate. Results from different samples were compared to a "control sample" corresponding to RNA prepared from one adult rat epithelium.
List of abbreviations used
Gene Expression Omnibus
olfactory sensory neuron
principal component analysis
polymerase chain reaction
RNA integrity number
The Centre National de la Recherche Scientifique, the Université de Rennes 1, the Conseil Régional de Bretagne and the Technical Support Working Group (TSWG) are thanked for their generous support and encouragement to FG. The authors are grateful to the referees for their constructive comments.
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