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Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques

Harrison, CN; Gale, RE; Linch, DC; (1998) Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques. LEUKEMIA , 12 (11) 1834 - 1839.

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Abstract

Most studies using X-chromosome inactivation patterns (XCIPs) to determine clonality in females have used polymorphic DNA loci, but interpretation may be complicated by the complexity of the differential methylation patterns necessary to distinguish active and inactive X-chromosomes. Recent description of polymorphisms within the transcribed region of three X-chromosome genes has enabled XCIP analysis of allele expression at the RNA level, which should circumvent this problem. We report here a quantitative RT-PCR assay for one of these loci, the iduronate-2-sulphatase (IDS) gene, using a mismatch primer to introduce a Bc/l cutting site in one of the alleles. DNA samples from 150 females were screened and 61 (41%) were found to be informative for the polymorphism. Reproducible results were obtained from clonal analysis of 56 RNA samples covering the complete spectrum of XCIP ratios. The values correlated closely with those obtained from the corresponding DNA samples analyzed using either PGK or HUMARA (r = 0.98) provided that the number of amplification cycles was limited to 25 to reduce heteroduplex formation. However, sample purity is of greater importance than in the DNA-based assays as contaminating red cells and platelets will still contain the relevant transcripts and could influence the result.

Type: Article
Title: Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques
Keywords: X-chromosome inactivation, IDS, RNA, HYPERVARIABLE PROBE M27-BETA, CLONAL ANALYSIS, METHYLATION PATTERNS, SAMPLES, SITES
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology
URI: http://discovery.ucl.ac.uk/id/eprint/33587
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