Protocol for the sequence analysis of ryanodine receptor subtype 1 gene transcripts from human leukocytes.
Background: The search for novel mutations in the ryanodine receptor subtype 1 (RYR1) gene causing malignant hyperthermia and central core disease is hampered by the fact that the gene contains 106 exons. Searching for novel mutations in complementary DNA (cDNA) requires an invasive muscle biopsy. A...
| Publicado en: | Anesthesiology Vol. 99; no. 2; pp. 289 - 297 |
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| Autores principales: | , , , |
| Formato: | research Journal Article |
| Publicado: |
Lippincott Williams & Wilkins
Aug2003
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=137955566&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 137955566 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00033022 1V8 jtl: Anesthesiology issn: 00033022 maglogo: N pubinfo: dt: Aug2003 vid: 99 iid: 2 pid: 433 pub: Lippincott Williams & Wilkins place: Baltimore, Maryland artinfo: ui: 137955566 137955566 NLM12883401 137955566 NLM12883401 137955566 ppf: 289 ppct: 8 formats: tig: atl: Protocol for the sequence analysis of ryanodine receptor subtype 1 gene transcripts from human leukocytes. aug: au: Kraev, Natasha Loke, Julian C P Kraev, Alexander MacLennan, David H affil: Department of Anaesthesia, University of Toronto, Ontario, Canada sug: subj: Calcium Leukocytes Metabolism DNA DNA Probes Documentation Reverse Transcriptase Polymerase Chain Reaction Polymorphism, Genetic Human Muscle, Skeletal Muscle, Skeletal Metabolism Calcium Metabolism RNA Genetic Techniques Nucleotides Validation Studies Comparative Studies Evaluation Research Multicenter Studies Clinical Assessment Tools ab: Background: The search for novel mutations in the ryanodine receptor subtype 1 (RYR1) gene causing malignant hyperthermia and central core disease is hampered by the fact that the gene contains 106 exons. Searching for novel mutations in complementary DNA (cDNA) requires an invasive muscle biopsy. Accordingly, an alternate source of RYR1 cDNA was sought for sequence analysis.Methods: Leukocytes were isolated from human blood and used for extraction of RNA and reverse transcription of messenger RNA into cDNA. A detailed protocol was developed in which overlapping fragments of RYR1 cDNA were amplified by polymerase chain reaction in a series of steps and used for double-strand sequencing.Results: The sequences of full-length leukocyte RYR1 cDNA obtained from four human blood samples were shown to be identical to the sequence of a human muscle RYR1 cDNA. The incidence of aberrant splicing was more pronounced in the blood-derived cDNAs, but this could be minimized by adequate sample preparation. Protocols to sequence alternatively spliced products were also developed. Several silent nucleotide polymorphisms were detected, and minor revisions were made to the RYR1 sequence.Conclusions: Because there are no differences in RYR1 transcript structure between muscle and leukocytes, aside from those that may be ascribed to RNA splicing aberrations during processing, leukocytes seem to be an adequate substitute tissue for screening the RYR1 gene for previously undiscovered mutations in families with malignant hyperthermia or central core disease. pubtype: Academic Journal doctype: research Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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