Pereira-Fantini Prue M, Rajapaksa Anushi E, Oakley Regina, Tingay David G
Neonatal Research Group, Murdoch Childrens Research Institute, Parkville, Australia.
Department of Paediatrics, University of Melbourne, Parkville, Australia.
Sci Rep. 2016 May 23;6:26476. doi: 10.1038/srep26476.
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.
早产儿常常需要侵入性支持，然而，即使是短时间不恰当地对肺部进行辅助通气也可能导致损伤。实时定量逆转录聚合酶链反应（qPCR）已广泛应用于通气诱导性肺损伤的研究，其中18S核糖体RNA（18S RNA）最常被用作内参基因。虽然这些研究的结果取决于所使用的内参基因的稳定性，但18S RNA的使用尚未得到验证。在本研究中，通过geNorm、Normfinder、Bestkeeper和ΔCt法等专用算法评估了五个候选内参基因（18S RNA、ACTB、GAPDH、TOP1和RPS29）在两个地理位置的表达谱，并确定了这些候选基因的整体稳定性（RefFinder）。二次研究考察了内参基因选择对两个经过充分验证的肺损伤标志物（EGR1和IL1B）相对表达的影响。在早产羔羊肺损伤模型中，RPS29内参基因的表达受组织位置影响；然而，我们确定个体通气策略会影响内参基因的稳定性。虽然18S RNA是早产羔羊肺研究中最常用的内参基因，但我们的结果表明GAPDH是更合适的候选基因。
