Identification of stable reference genes in peripheral blood mononuclear cells from type 2 diabetes mellitus patients

Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring r...

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Published inScientific reports Vol. 13; no. 1; pp. 486 - 12
Main Authors Hazarika, Ankita, Nongkhlaw, Bajanai, Mukhopadhyay, Arpita
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 10.01.2023
Nature Publishing Group
Nature Portfolio
Subjects
631/337/2019
692/699/2743/137/773
Actin
Diabetes
Diabetes mellitus (non-insulin dependent)
Diabetes Mellitus, Type 2 - genetics
Female
Gene expression
Gene Expression Profiling
Glyceraldehyde-3-phosphate dehydrogenase
Humanities and Social Sciences
Humans
Leukocytes, Mononuclear
Male
multidisciplinary
Peptidylprolyl isomerase
Peripheral blood mononuclear cells
Polymerase chain reaction
Real-Time Polymerase Chain Reaction
Reference Standards
Reproducibility of Results
RNA, Messenger - genetics
Science
Science (multidisciplinary)
Tryptophan
Tryptophan 5-monooxygenase
Online AccessGet full text
ISSN2045-2322
2045-2322
DOI10.1038/s41598-023-27460-3

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Abstract Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB ; Peptidylprolyl Isomerase B, PPIB ; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH ); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [ GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P  = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.
AbstractList Abstract Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.
Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB ; Peptidylprolyl Isomerase B, PPIB ; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH ); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [ GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P  = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.
Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.
Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.
ArticleNumber 486
Author Hazarika, Ankita
Nongkhlaw, Bajanai
Mukhopadhyay, Arpita
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  fullname: Hazarika, Ankita
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  givenname: Bajanai
  surname: Nongkhlaw
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  surname: Mukhopadhyay
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  organization: Division of Nutrition, St. John’s Research Institute, St. John’s National Academy of Health Sciences
BackLink https://www.ncbi.nlm.nih.gov/pubmed/36627346$$D View this record in MEDLINE/PubMed
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Snippet Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain...
Abstract Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase...
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StartPage 486
SubjectTerms 631/337/2019
692/699/2743/137/773
Actin
Diabetes
Diabetes mellitus (non-insulin dependent)
Diabetes Mellitus, Type 2 - genetics
Female
Gene expression
Gene Expression Profiling
Glyceraldehyde-3-phosphate dehydrogenase
Humanities and Social Sciences
Humans
Leukocytes, Mononuclear
Male
multidisciplinary
Peptidylprolyl isomerase
Peripheral blood mononuclear cells
Polymerase chain reaction
Real-Time Polymerase Chain Reaction
Reference Standards
Reproducibility of Results
RNA, Messenger - genetics
Science
Science (multidisciplinary)
Tryptophan
Tryptophan 5-monooxygenase
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Title Identification of stable reference genes in peripheral blood mononuclear cells from type 2 diabetes mellitus patients
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