Dataset Information

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 ?Ct 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.

SUBMITTER: Pereira-Fantini PM

PROVIDER: S-EPMC4876477 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Pereira-Fantini Prue M PM Rajapaksa Anushi E AE Oakley Regina R Tingay David G DG

Scientific reports 20160523

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 us ...[more]

PMID: 27210246

Similar Datasets

Project description:BackgroundLung ultrasound (LUS) may not detect small, dynamic changes in lung volume. Mean greyscale measurement using computer-assisted image analysis (Q-LUSMGV) may improve the precision of these measurements.MethodsPreterm lambs (n = 40) underwent LUS of the dependent or non-dependent lung during static pressure-volume curve mapping. Total and regional lung volumes were determined using the super-syringe technique and electrical impedance tomography. Q-LUSMGV and gold standard measurements of lung volume were compared in 520 images.ResultsDependent Q-LUSMGV moderately correlated with total lung volume (rho = 0.60, 95% CI 0.51-0.67) and fairly with right whole (rho = 0.39, 0.27-0.49), central (rho = 0.38, 0.27-0.48), ventral (rho = 0.41, 0.31-0.51) and dorsal regional lung volumes (rho = 0.32, 0.21-0.43). Non-dependent Q-LUSMGV moderately correlated with total lung volume (rho = 0.57, 0.48-0.65) and fairly with right whole (rho = 0.43, 0.32-0.52), central (rho = 0.46, 0.35-0.55), ventral (rho = 0.36, 0.25-0.47) and dorsal lung volumes (rho = 0.36, 0.25-0.47). All correlation coefficients were statistically significant. Distinct inflation and deflation limbs, and sonographic pulmonary hysteresis occurred in 95% of lambs. The greatest changes in Q-LUSMGV occurred at the opening and closing pressures.ConclusionQ-LUSMGV detected changes in total and regional lung volume and offers objective quantification of LUS images, and may improve bedside discrimination of real-time changes in lung volume.ImpactLung ultrasound (LUS) offers continuous, radiation-free imaging that may play a role in assessing lung recruitment but may not detect small changes in lung volume. Mean greyscale image analysis using computer-assisted quantitative LUS (Q-LUSMGV) moderately correlated with changes in total and regional lung volume. Q-LUSMGV identified opening and closing pressure and pulmonary hysteresis in 95% of lambs. Computer-assisted image analysis may enhance LUS estimation of lung recruitment at the bedside. Future research should focus on improving precision prior to clinical translation.

Project description:MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets