Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults
Abstract Background Preterm birth is the leading cause of neonatal morbidity and mortality, but research efforts in neonatology are complicated due to the unavailability of large volume blood samples. Whole blood assays can be used to overcome this problem by performing both functional and gene expr...
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doaj-995702a8ae8742e38bf3955ae1a99a772021-07-04T11:33:33ZengBMCBMC Genomics1471-21642021-06-0122111210.1186/s12864-021-07801-0Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adultsKristin Hieronymus0Benjamin Dorschner1Felix Schulze2Neeta L. Vora3Joel S. Parker4Jennifer Lucia Winkler5Angela Rösen-Wolff6Stefan Winkler7Department of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität DresdenDepartment of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität DresdenDepartment of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität DresdenDepartment of Obstetrics and Gynecology, University of North Carolina School of MedicineDepartment of Genetics, Lineberger Comprehensive Cancer Center, University of North CarolinaDepartment of Gynecology and Obstetrics, University Hospital Carl Gustav Carus, Technische Universität DresdenDepartment of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität DresdenDepartment of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität DresdenAbstract Background Preterm birth is the leading cause of neonatal morbidity and mortality, but research efforts in neonatology are complicated due to the unavailability of large volume blood samples. Whole blood assays can be used to overcome this problem by performing both functional and gene expression studies using small amounts of blood. Gene expression studies using RT-qPCR estimate mRNA-levels of target genes normalized to reference genes. The goal of this study was to identify and validate stable reference genes applicable to cord blood samples obtained from developing neonates of different gestational age groups as well as to adult peripheral blood samples. Eight reference gene candidates (ACTB, B2M, GAPDH, GUSB, HPRT, PPIB, RPLP0, RPL13) were analyzed using the three published software algorithms Bestkeeper, GeNorm and NormFinder. Results A normalization factor consisting of ACTB and PPIB allows for comparative expression analyses of neonatal samples from different gestational age groups. Normalization factors consisting of GAPDH and PPIB or ACTB and GAPDH are suitable when samples from preterm and full-term neonates and adults are compared. However, all candidate reference genes except RPLP0 exhibited significant intergroup gene expression variance and a higher gene expression towards an older age which resulted in a small but statistically significant systematic bias. Systematic analysis of RNA-seq data revealed new reference gene candidates with potentially superior stability. Conclusions The current study identified suitable normalization factors and proposed the use of the additional single gene RPLP0 to avoid systematic bias. This combination will enable comparative analyses not only between neonates of different gestational ages, but also between neonates and adults, as it facilitates more detailed investigations of developmental gene expression changes. The use of software algorithms did not prevent unintended systematic bias. This generally highlights the need for careful validation of such results to prevent false interpretation of potential age-dependent changes in gene expression. To identify the most stable reference genes in the future, RNA-seq based global approaches are recommended.https://doi.org/10.1186/s12864-021-07801-0Housekeeping genesComparative gene expression analysisValidationWhole bloodNeonatesInfants |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kristin Hieronymus Benjamin Dorschner Felix Schulze Neeta L. Vora Joel S. Parker Jennifer Lucia Winkler Angela Rösen-Wolff Stefan Winkler |
spellingShingle |
Kristin Hieronymus Benjamin Dorschner Felix Schulze Neeta L. Vora Joel S. Parker Jennifer Lucia Winkler Angela Rösen-Wolff Stefan Winkler Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults BMC Genomics Housekeeping genes Comparative gene expression analysis Validation Whole blood Neonates Infants |
author_facet |
Kristin Hieronymus Benjamin Dorschner Felix Schulze Neeta L. Vora Joel S. Parker Jennifer Lucia Winkler Angela Rösen-Wolff Stefan Winkler |
author_sort |
Kristin Hieronymus |
title |
Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
title_short |
Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
title_full |
Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
title_fullStr |
Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
title_full_unstemmed |
Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
title_sort |
validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults |
publisher |
BMC |
series |
BMC Genomics |
issn |
1471-2164 |
publishDate |
2021-06-01 |
description |
Abstract Background Preterm birth is the leading cause of neonatal morbidity and mortality, but research efforts in neonatology are complicated due to the unavailability of large volume blood samples. Whole blood assays can be used to overcome this problem by performing both functional and gene expression studies using small amounts of blood. Gene expression studies using RT-qPCR estimate mRNA-levels of target genes normalized to reference genes. The goal of this study was to identify and validate stable reference genes applicable to cord blood samples obtained from developing neonates of different gestational age groups as well as to adult peripheral blood samples. Eight reference gene candidates (ACTB, B2M, GAPDH, GUSB, HPRT, PPIB, RPLP0, RPL13) were analyzed using the three published software algorithms Bestkeeper, GeNorm and NormFinder. Results A normalization factor consisting of ACTB and PPIB allows for comparative expression analyses of neonatal samples from different gestational age groups. Normalization factors consisting of GAPDH and PPIB or ACTB and GAPDH are suitable when samples from preterm and full-term neonates and adults are compared. However, all candidate reference genes except RPLP0 exhibited significant intergroup gene expression variance and a higher gene expression towards an older age which resulted in a small but statistically significant systematic bias. Systematic analysis of RNA-seq data revealed new reference gene candidates with potentially superior stability. Conclusions The current study identified suitable normalization factors and proposed the use of the additional single gene RPLP0 to avoid systematic bias. This combination will enable comparative analyses not only between neonates of different gestational ages, but also between neonates and adults, as it facilitates more detailed investigations of developmental gene expression changes. The use of software algorithms did not prevent unintended systematic bias. This generally highlights the need for careful validation of such results to prevent false interpretation of potential age-dependent changes in gene expression. To identify the most stable reference genes in the future, RNA-seq based global approaches are recommended. |
topic |
Housekeeping genes Comparative gene expression analysis Validation Whole blood Neonates Infants |
url |
https://doi.org/10.1186/s12864-021-07801-0 |
work_keys_str_mv |
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