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1.
Genomics ; 90(4): 482-92, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17706913

ABSTRACT

We originally showed that the protocadherin 15 gene (Pcdh15) is necessary for hearing and balance functions; mutations in Pcdh15 affect hair cell development in Ames waltzer (av) mice. Here we extend that study to understand better how Pcdh15 operates in a cell. The original report identified 33 exons in Pcdh15, with exon 1 being noncoding; additional exons of Pcdh15 have since been reported. The 33 exons of Pcdh15 described originally are embedded in 409 kb of mouse genomic sequence, while the corresponding exons of human PCDH15 are spread over 980 kb of genomic DNA; the exons in Pcdh15/PCDH15 range in size from 9 to approximately 2000 bp. The genomic organization of Pcdh15/PCDH15 bears similarity to that of cadherin 23, but differs significantly from other protocadherin genes, such as Pcdhalpha, beta, or gamma. A CpG island is located approximately 2900 bp upstream of the PCDH15 transcriptional start site. The Pcdh15/PCDH15 promoter lacks TATAA or CAAT sequences within 100 bases upstream of the transcription start site; deletion mapping showed that Pcdh15 harbors suppressor and enhancer elements. Preliminary searches for alternatively spliced transcripts of Pcdh15 identified novel splice variants not reported previously. Results from our study show that both mouse and human protocadherin 15 genes have complex genomic structures and transcription control mechanisms.


Subject(s)
Alternative Splicing , Cadherins/genetics , Promoter Regions, Genetic , 3T3-L1 Cells , Amino Acid Sequence , Animals , Animals, Newborn , Cadherin Related Proteins , Chromosome Mapping , CpG Islands , Humans , Mice , Molecular Sequence Data , Protein Isoforms/genetics , Sequence Analysis, DNA
2.
Otol Neurotol ; 28(1): 116-23, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16983313

ABSTRACT

HYPOTHESIS: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. BACKGROUND: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. METHODS: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. RESULTS: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. CONCLUSION: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.


Subject(s)
RNA/genetics , RNA/metabolism , Temporal Bone/metabolism , Temporal Bone/pathology , Animals , Biological Specimen Banks , DNA Primers/genetics , DNA, Complementary/genetics , Ear, Inner/metabolism , Ear, Inner/pathology , Endolymphatic Hydrops/genetics , Endolymphatic Hydrops/metabolism , Endolymphatic Hydrops/pathology , Excitatory Amino Acid Transporter 1/genetics , Gene Expression/genetics , Glutamic Acid/genetics , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/genetics , Guinea Pigs , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
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