Optimal Reference Gene Selection for Expression Studies in Human Reticulocytes
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved..
Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports. Real-time quantitative PCR was performed on reticulocytes in 20 healthy volunteers, 15 HS patients, and 10 βTI patients. Threshold cycle values were compared with fold-change method and RefFinder software. The stable reference genes recommended by RefFinder were validated with SLC4A1 and flow cytometric eosin-5'-maleimide binding assay values in HS patients and HBG2 and high performance liquid chromatography-derived percentage of hemoglobin F in βTI. Comprehensive ranking predicted MPP1 and GAPDH as optimal reference genes for reticulocytes that were not affected in HS and βTI. This was further confirmed on validation with eosin-5'-maleimide results and percentage of hemoglobin F in HS and βTI patients, respectively. Hence, MPP1 and GAPDH are good reference genes for reticulocyte expression studies compared with ACTB and RN18S1, the two most commonly used reference genes.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2018 |
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| Erschienen: |
2018 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:20 |
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| Enthalten in: |
The Journal of molecular diagnostics : JMD - 20(2018), 3 vom: 21. Mai, Seite 326-333 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Aggarwal, Anu [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 16.09.2019 Date Revised 08.04.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jmoldx.2018.01.009 |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM281330840 |
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| 520 | |a Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports. Real-time quantitative PCR was performed on reticulocytes in 20 healthy volunteers, 15 HS patients, and 10 βTI patients. Threshold cycle values were compared with fold-change method and RefFinder software. The stable reference genes recommended by RefFinder were validated with SLC4A1 and flow cytometric eosin-5'-maleimide binding assay values in HS patients and HBG2 and high performance liquid chromatography-derived percentage of hemoglobin F in βTI. Comprehensive ranking predicted MPP1 and GAPDH as optimal reference genes for reticulocytes that were not affected in HS and βTI. This was further confirmed on validation with eosin-5'-maleimide results and percentage of hemoglobin F in HS and βTI patients, respectively. Hence, MPP1 and GAPDH are good reference genes for reticulocyte expression studies compared with ACTB and RN18S1, the two most commonly used reference genes | ||
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| 700 | 1 | |a Viswanathan, Ganesh K |e verfasserin |4 aut | |
| 700 | 1 | |a Sharma, Prashant |e verfasserin |4 aut | |
| 700 | 1 | |a Sachdeva, ManUpdesh S |e verfasserin |4 aut | |
| 700 | 1 | |a Bansal, Deepak |e verfasserin |4 aut | |
| 700 | 1 | |a Malhotra, Pankaj |e verfasserin |4 aut | |
| 700 | 1 | |a Das, Reena |e verfasserin |4 aut | |
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