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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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12864-021-07801-0.

bmc genomics

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