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Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3
(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular de...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460478/ https://www.ncbi.nlm.nih.gov/pubmed/32823754 http://dx.doi.org/10.3390/diagnostics10080588 |
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author | Wongfieng, Wipaporn Nutalai, Rungtiwa Jumnainsong, Amonrat Leelayuwat, Chanvit |
author_facet | Wongfieng, Wipaporn Nutalai, Rungtiwa Jumnainsong, Amonrat Leelayuwat, Chanvit |
author_sort | Wongfieng, Wipaporn |
collection | PubMed |
description | (1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 10(7) copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R(2) = 0.995, KIR3DL3; R(2) = 0.996, but lower sensitivity of 10(3) copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications. |
format | Online Article Text |
id | pubmed-7460478 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-74604782020-09-03 Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 Wongfieng, Wipaporn Nutalai, Rungtiwa Jumnainsong, Amonrat Leelayuwat, Chanvit Diagnostics (Basel) Article (1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 10(7) copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R(2) = 0.995, KIR3DL3; R(2) = 0.996, but lower sensitivity of 10(3) copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications. MDPI 2020-08-13 /pmc/articles/PMC7460478/ /pubmed/32823754 http://dx.doi.org/10.3390/diagnostics10080588 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Wongfieng, Wipaporn Nutalai, Rungtiwa Jumnainsong, Amonrat Leelayuwat, Chanvit Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title | Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title_full | Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title_fullStr | Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title_full_unstemmed | Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title_short | Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3 |
title_sort | quantitative multiplex real-time reverse transcriptase–polymerase chain reaction with fluorescent probe detection of killer immunoglobulin-like receptors, kir2dl4/3dl3 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460478/ https://www.ncbi.nlm.nih.gov/pubmed/32823754 http://dx.doi.org/10.3390/diagnostics10080588 |
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