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NanoCAGE analysis of the mouse olfactory epithelium identifies the expression of vomeronasal receptors and of proximal LINE elements.

Pascarella G, Lazarevic D, Plessy C, Bertin N, Akalin A, Vlachouli C, Simone R, Faulkner GJ, Zucchelli S, Kawai J, Daub CO, Hayashizaki Y, Lenhard B, Carninci P, Gustincich S - Front Cell Neurosci (2014)

Bottom Line: These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs).We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR.This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression.

View Article: PubMed Central - PubMed

Affiliation: Area of Neuroscience, International School for Advanced Studies (SISSA) Trieste, Italy ; RIKEN Yokohama Institute, Center for Life Science Technologies, Division of Genomic Technologies Tsurumi-ku, Yokohama, Japan.

ABSTRACT
By coupling laser capture microdissection to nanoCAGE technology and next-generation sequencing we have identified the genome-wide collection of active promoters in the mouse Main Olfactory Epithelium (MOE). Transcription start sites (TSSs) for the large majority of Olfactory Receptors (ORs) have been previously mapped increasing our understanding of their promoter architecture. Here we show that in our nanoCAGE libraries of the mouse MOE we detect a large number of tags mapped in loci hosting Type-1 and Type-2 Vomeronasal Receptors genes (V1Rs and V2Rs). These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs). We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR. This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression.

No MeSH data available.


Related in: MedlinePlus

Validation of nanoCAGE data by qRT-PCR confirms that the expression levels of selected VRs in the MOE of young and adult mice are comparable to the expression levels of ORs genes with similar tag counts in nanoCAGE libraries. The qRT-PCR validation was performed in triplicates on RNA purified from the dissected MOE and VNO of P21 (males n = 5, females n = 5) and P50 (males n = 5, females n = 5) C57BL/6J mice. All primers used were designed in an exon-spanning fashion; the Ct values of each target were normalized on Gapdh Ct values. The expression levels in the VNO and the copy number calculation are shown in Supplementary Figure S3.
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Figure 6: Validation of nanoCAGE data by qRT-PCR confirms that the expression levels of selected VRs in the MOE of young and adult mice are comparable to the expression levels of ORs genes with similar tag counts in nanoCAGE libraries. The qRT-PCR validation was performed in triplicates on RNA purified from the dissected MOE and VNO of P21 (males n = 5, females n = 5) and P50 (males n = 5, females n = 5) C57BL/6J mice. All primers used were designed in an exon-spanning fashion; the Ct values of each target were normalized on Gapdh Ct values. The expression levels in the VNO and the copy number calculation are shown in Supplementary Figure S3.

Mentions: In order to confirm the reliability of nanoCAGE in detecting the expression of selected VRs genes we performed a real-time quantitative RT-PCR (qRT-PCR) on OMP (TPM = 157), an OR gene with an high expression level (Olfr 110, TPM = 22.7), three vomeronasal receptor genes (Vmn1r201, TPM = 0.18; Vmn2r26, TPM = 0.15; Vmn2r69, TPM = 0.22) and three OR genes with a low tag count comparable to the selected vomeronasal receptor targets (Olfr480, TPM = 0.25; Olfr995, TPM = 0.22; Olfr1413, TPM = 0.16) (Figure 6). qRT-PCR reactions were performed in triplicates on total RNA purified from dissections of the MOE and VNO of P21 (males n = 5 and females n = 5) and P50 (males n = 5 and females n = 5) C57BL/6J animals. As a negative control we performed qRT-PCR on the same targets using as a template total RNA purified from mouse liver. After copy numbers count and normalization with Gapdh Ct values, we were able to quantify mRNAs for Olfr995, Olfr480, Olfr1413, Vmn2r26, and Vmn2r69 and to confirm that qRT-PCR data are consistent with the expression levels detected by nanoCAGE (Supplementary Figure S3). We were not able to detect the expression of Vmn1r201 in P50 female mice. As expected, we were not able to amplify any of the targets from liver RNA apart from Gapdh. We detected expression of Olfr110, Olfr995, and Olfr1413 genes also in the VNO samples.


NanoCAGE analysis of the mouse olfactory epithelium identifies the expression of vomeronasal receptors and of proximal LINE elements.

Pascarella G, Lazarevic D, Plessy C, Bertin N, Akalin A, Vlachouli C, Simone R, Faulkner GJ, Zucchelli S, Kawai J, Daub CO, Hayashizaki Y, Lenhard B, Carninci P, Gustincich S - Front Cell Neurosci (2014)

Validation of nanoCAGE data by qRT-PCR confirms that the expression levels of selected VRs in the MOE of young and adult mice are comparable to the expression levels of ORs genes with similar tag counts in nanoCAGE libraries. The qRT-PCR validation was performed in triplicates on RNA purified from the dissected MOE and VNO of P21 (males n = 5, females n = 5) and P50 (males n = 5, females n = 5) C57BL/6J mice. All primers used were designed in an exon-spanning fashion; the Ct values of each target were normalized on Gapdh Ct values. The expression levels in the VNO and the copy number calculation are shown in Supplementary Figure S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927265&req=5

Figure 6: Validation of nanoCAGE data by qRT-PCR confirms that the expression levels of selected VRs in the MOE of young and adult mice are comparable to the expression levels of ORs genes with similar tag counts in nanoCAGE libraries. The qRT-PCR validation was performed in triplicates on RNA purified from the dissected MOE and VNO of P21 (males n = 5, females n = 5) and P50 (males n = 5, females n = 5) C57BL/6J mice. All primers used were designed in an exon-spanning fashion; the Ct values of each target were normalized on Gapdh Ct values. The expression levels in the VNO and the copy number calculation are shown in Supplementary Figure S3.
Mentions: In order to confirm the reliability of nanoCAGE in detecting the expression of selected VRs genes we performed a real-time quantitative RT-PCR (qRT-PCR) on OMP (TPM = 157), an OR gene with an high expression level (Olfr 110, TPM = 22.7), three vomeronasal receptor genes (Vmn1r201, TPM = 0.18; Vmn2r26, TPM = 0.15; Vmn2r69, TPM = 0.22) and three OR genes with a low tag count comparable to the selected vomeronasal receptor targets (Olfr480, TPM = 0.25; Olfr995, TPM = 0.22; Olfr1413, TPM = 0.16) (Figure 6). qRT-PCR reactions were performed in triplicates on total RNA purified from dissections of the MOE and VNO of P21 (males n = 5 and females n = 5) and P50 (males n = 5 and females n = 5) C57BL/6J animals. As a negative control we performed qRT-PCR on the same targets using as a template total RNA purified from mouse liver. After copy numbers count and normalization with Gapdh Ct values, we were able to quantify mRNAs for Olfr995, Olfr480, Olfr1413, Vmn2r26, and Vmn2r69 and to confirm that qRT-PCR data are consistent with the expression levels detected by nanoCAGE (Supplementary Figure S3). We were not able to detect the expression of Vmn1r201 in P50 female mice. As expected, we were not able to amplify any of the targets from liver RNA apart from Gapdh. We detected expression of Olfr110, Olfr995, and Olfr1413 genes also in the VNO samples.

Bottom Line: These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs).We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR.This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression.

View Article: PubMed Central - PubMed

Affiliation: Area of Neuroscience, International School for Advanced Studies (SISSA) Trieste, Italy ; RIKEN Yokohama Institute, Center for Life Science Technologies, Division of Genomic Technologies Tsurumi-ku, Yokohama, Japan.

ABSTRACT
By coupling laser capture microdissection to nanoCAGE technology and next-generation sequencing we have identified the genome-wide collection of active promoters in the mouse Main Olfactory Epithelium (MOE). Transcription start sites (TSSs) for the large majority of Olfactory Receptors (ORs) have been previously mapped increasing our understanding of their promoter architecture. Here we show that in our nanoCAGE libraries of the mouse MOE we detect a large number of tags mapped in loci hosting Type-1 and Type-2 Vomeronasal Receptors genes (V1Rs and V2Rs). These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs). We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR. This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression.

No MeSH data available.


Related in: MedlinePlus