Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model

Abstract

Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate.

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

“…Finally, the different results obtained for TNFα expression levels between groups (resistant and susceptible to H. contortus infection) depending on the reference gene used have demonstrated that inappropriate reference gene selection can lead to misinterpretation of gene expression results. This fact is due to high expression variations in a non-stable reference gene being transferred to the target-gene, which can lead to under-or over-estimation of the relative quantification data and important changes in statistical significance, corroborating the findings of other studies [20,33,34,45].…”

“…Selection of the reference gene candidates was based on those commonly used for RT-qPCR studies in different ruminant tissues [30][31][32][33][34]. Five reference gene candidates with different cellular functions were selected: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glucose 6-phosphate dehydrogenase (G6PDH), enzymes associated with carbohydrate metabolism; tyrosine 3-monoxygenase (YWHAZ), involved in intercellular signal transduction; actin beta (ACTB), cytoskeletal structural protein; beta-2 microglobulin (B2M), a component of histocompatibility complex (MHC) class I beta chain.…”

Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction

“…Thus, it is advisable to evaluate reference genes by more than one method and integrate the data to generate final ranking, especially in tissues like liver. Here, we calculated the comprehensive ranking by geometric mean of results by all the software programs324041. And validation by Leptin and Ppar-γ expression assessment confirmed the reliability of this strategy.…”

“…On the other hand, the least stable genes were Gapdh > Hmbs > Pgk1 showed by geNorm, Gapdh > Hmbs > Rpl13a by NormFinder and Gapdh > Pgk1 > Hmbs by BestKeeper (Table 3). While reference genes ranking varied slightly by algorithm, the overall ranking of the best candidate reference genes were calculated through geometric means of the three ranking numbers and the most stable reference genes had the smallest geometric mean324041 (Table 3). The comprehensive ranking showed that β2m and Hprt appear to be the most suitable reference gene pair in iWAT of CR.…”

“…There were some differences in the ranking of reference genes between algorithms. However, the comprehensive ranking based on the geometric mean of the ranks by these algorithms324041 showed that Ppia is the most stable gene and Gapdh is the least stable one.…”