Computational protocol: Long-Distance Retinoid Signaling in the Zebra Finch Brain

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Protocol publication

[…] A cDNA from the ESTIMA database (Songbird Neurogenomics Initiative; was available for RXRα and was used for expression analysis. For all other genes examined, no clones were available in ESTIMA. We therefore PCR-cloned fragments corresponding to their zebra finch orthologs. For this purpose, we first isolated total brain RNA from an adult male zebra finch using the TRIzol method. We then used reverse transcriptase SuperScript II (Invitrogen) to transcribe total brain RNA, synthesized second strands using T4 DNA polymerase, digested RNA with RNAse H, and used the first-strand cDNA directly as a PCR template with gene specific primers. We designed primer pairs for RXRγ and CYP26A1 based on the genomic sequence, aided by examination of conserved regions identified through the alignment of the orthologs from several species deposited in GenBank, if genomic sequence quality was poor. The primer sequences were as follows: For RXRγ, forward 5′-GGGAAGCACTATGGGGTGTA-3′, reverse 5′-CTGATCGACAAGCGCCAGCG-3′, predicted fragment length 799 bp. For CYP26A1: forward 5′-CTGAATGAGTCTGCCACAG-3′, reverse 5′-CTTCATGTCTCCATCTCCAG-3′, predicted fragment length 406 bp. For CYP26B1 and CYP26C1, primer sequences for cloning were based on primer sequences previously used for chicken CYP26B1 and CYP26C1 , . All PCR products were examined on agarose gels, purified with the Qiaquick PCR purification kit (Qiagen) and inserted into pGEM-T easy vectors (Promega); resulting clones were sequenced for identification, and full-length ORF sequences were deposited in GenBank under the following accession numbers: RXRα: HQ830555; RXRγ: HQ830557; Cyp26A1: HQ830558; Cyp26B1: HQ830556; Cyp26C1: HQ830559 (for Cyp26C1, only a partial ORF sequence was cloned). [...] ESTIMA zebra finch clone SB03015A2E11.f1 (GenBank accession #: DV949832) corresponds to the 3′ portion of the RXRα mRNA, extending from the middle of the coding region for the ligand-binding domain to the polyA tail (.A). To assess the likelihood of cross-hybridization between this RXRα clone and other RXRs, we blat aligned the RXRα clone sequence against the entire zebra finch genome (UCSC browser). Whereas the EST sequence (514bp) fully aligns with the RXRα locus with 99.7% identity (blat score 1358), it has only a very short partial alignment (88bp) at the RXRγ locus at 89.7% similarity (blat score 71). Probe specificity for RXRα as opposed to RXRγ was further indicated by a total lack of overlap between the two expression patterns. […]

Pipeline specifications

Software tools ESTIMA, BLAT
Application Transcription analysis
Organisms Taeniopygia guttata
Chemicals Tretinoin, Vitamin A