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.
Highlights
Preterm birth is associated with developmental immaturity of the lung
18S ribosomal RNA (18S RNA) is the most commonly described reference in preterm lamb models of lung injury[7,8,9,10,11,12,13,14], ACTB and GAPDH had been previously validated in adult sheep lung tissue[19], RPS29 has been extensively used and validated in small animal models[20,21] and TOP1 studied in a rabbit respiratory model[22]
The preterm lamb model of lung injury is complex, with reference gene expression potentially influenced by tissue sample location, ventilation strategy, maternal factors and other adjunctive therapies, such as exogenous surfactant and antenatal corticosteroids
Summary
Preterm birth is associated with developmental immaturity of the lung. Functionally this limits the ability of the lung to maintain effective aeration and gas exchange. Even brief periods of assisted ventilation applied inappropriately to the developmentally and mechanically fragile preterm lung can initiate inflammation and injury[2,3,4], via a variety of different mechanisms throughout the lung, which create a gravity-dependent heterogeneous pattern of injury throughout the lung, and influence the benefit of subsequent therapies such as surfactant[5,6]. Evaluation of five reference genes was performed on two geographical and gravity-dependent distinct areas of lung and obtained following a variety of assisted ventilation strategies, by analyzing reference gene stability with three different algorithms (geNorm[15], NormFinder[16] and BestKeeper[17]). Relative gene expression of Early growth response protein 1 (EGR1), a commonly employed injury biomarker for VILI, and interleukin 1 beta (IL1B), an early marker of inflammation in preterm lambs and other models of lung injury[1,6,7,18], when normalized against the most commonly employed reference gene in VILI studies (18S RNA), a consistently high ranked reference gene candidate (Glyceraldehyde 3-phosphate dehydrogenase; GAPDH), and the worst reference gene candidate (Ribosomal Protein S29; RPS29)
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