Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis

Kerékgyártó, Márta; Németh, Nóra; Kerekes, Tamás; Rónai, Zsolt; Guttman, András

doi:10.1016/j.chroma.2013.02.062

Cited by 11 publications

(7 citation statements)

References 33 publications

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“…Accuracy of fragment size determination of the CE technique was 80–85% by utilizing the co‐injection of a 20 and a 5000 bp‐long alignment marker with each sample, providing unambiguous identification of the two isoforms. Separations were completed in less than 3 min, and no subsequent steps (such as densitometry of each band in combination with submarine gel electrophoresis) were necessary as the concentration of the fragments were simply determined based on peak areas by the Q‐Expert software .…”

Section: Resultsmentioning

confidence: 99%

“…A miniaturized CE system utilizing LED‐induced fluorescence (LEDIF) was already introduced by our group in the previous issue of this Miniaturization Series . It was also used for large‐scale analysis in laboratory tests and clinical settings to investigate two adjacent putative mRNA‐binding SNPs ( rs1046322A and rs9457C ) in wolframin (WFS1) gene .…”

Section: Introductionmentioning

confidence: 99%

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Rapid identification of human SNAP‐25 transcript variants by a miniaturized capillary electrophoresis system

et al. 2013

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The 25 kDa synaptosomal-associated protein (SNAP-25) is a crucial component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and plays an important role in neurotransmission in the central nervous system. SNAP-25 has two different splice variants, SNAP-25a and SNAP-25b, differing in nine amino acids that results in a slight functional alteration of the generated soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. Two independent techniques, a PCR-miniaturized CE method and a real-time PCR based approach were elaborated for the specific and quantitative detection of the two SNAP-25 transcription variants. DNA-constructs coding for the two isoforms were used for optimization. Excellent specificity was observed with the use of our previously described highly sensitive miniaturized CE system in combination with quantitative PCR. The ratio of the two isoforms were reliably detected in a range of at least four orders of magnitude with a linear regression of R(2) = 0.987. Expression of the two isoforms was determined in human samples, where SNAP-25 was detected even in non-neural tissues, although at approximately a 100-fold lower level compared to the central nervous system. The relative amount of the SNAP-25b isoform was higher in the brain, whereas expression of SNAP-25a variant proved to be slightly higher in extra-neural cell types. The genomics approach in conjunction with the miniaturized CE system introduced in this paper is readily applicable for rapid alternative splice variant analysis.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid identification of human SNAP‐25 transcript variants by a miniaturized capillary electrophoresis system

et al. 2013

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“…A pen-shaped capillary cartridge system (Figure 7) using LEDIF detection has been recently described for rapid haplotyping of putative microRNA-binding sites in a gene associated with type 2 diabetes mellitus (167). With electrokinetic injection, an LOD of 2 ng/ml was possible.…”

Section: Other Capillary Scale Fluorescence/chemiluminescence Detectorsmentioning

confidence: 99%

Light-Emitting Diodes for Analytical Chemistry

Macka

Piasecki

Dasgupta

2014

Annual Rev. Anal. Chem.

114

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Light-emitting diodes (LEDs) are playing increasingly important roles in analytical chemistry, from the final analysis stage to photoreactors for analyte conversion to actual fabrication of and incorporation in microdevices for analytical use. The extremely fast turn-on/off rates of LEDs have made possible simple approaches to fluorescence lifetime measurement. Although they are increasingly being used as detectors, their wavelength selectivity as detectors has rarely been exploited. From their first proposed use for absorbance measurement in 1970, LEDs have been used in analytical chemistry in too many ways to make a comprehensive review possible. Hence, we critically review here the more recent literature on their use in optical detection and measurement systems. Cloudy as our crystal ball may be, we express our views on the future applications of LEDs in analytical chemistry: The horizon will certainly become wider as LEDs in the deep UV with sufficient intensity become available.

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“…Ultrathin‐layer gel electrophoresis was shown as an automated and efficient alternative to the traditional method allowing rapid and sensitive analysis of PCR products . CE as well as its miniaturized alternatives, such as microchip electrophoresis have also been reportedly used in SNP studies as they offer automated and highly sensitive detection of DNA fragments. Moreover, CE offers further advantages.…”

Section: Introductionmentioning

confidence: 99%

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene

et al. 2016

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The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

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Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis

Cited by 11 publications

References 33 publications

Rapid identification of human SNAP‐25 transcript variants by a miniaturized capillary electrophoresis system

Rapid identification of human SNAP‐25 transcript variants by a miniaturized capillary electrophoresis system

Light-Emitting Diodes for Analytical Chemistry

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene

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