The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time.

Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.

Modulation of Lgl1 by steroid, retinoic acid, and vitamin D models complex transcriptional regulation during alveolarization.

Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA), and vitamin D (VitD). Bronchopulmonary dysplasia, a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of bronchopulmonary dysplasia, reduced levels of late gestation lung 1 (Lgl1) normalize during recovery. We show that GC (100 nM) stimulates (7- to 115-fold) and VitD (100 microM) suppresses (twofold) Lgl1 expression. RA (all-trans/9-cis, 10 microM) effects are biphasic. From postnatal days 7-10, RA was stimulatory (twofold) at 24 h, after which effects were inhibitory (3- to 15-fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared with 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.

Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice.

BACKGROUND: Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. METHODS: An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation. RESULTS: Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics. CONCLUSION: Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.

Lgl1 is suppressed in oxygen toxicity animal models of bronchopulmonary dysplasia and normalizes during recovery in air.

Bronchopulmonary dysplasia (BPD), a major cause of morbidity in premature infants, is characterized by arrest of lung growth and inhibited alveologenesis. We had earlier cloned late-gestation lung 1 (LGL1), a glucocorticoid (GC)-induced, developmentally regulated gene in lung mesenchyme, and showed that reduced levels of late-gestation lung 1 protein (lgl1) inhibit lung branching. Maximal fetal expression of LGL1 is concordant with the onset of alveolar septation, suggesting an additional role for lgl1 in alveologenesis. At postnatal d 7, during the period of maximal septation in postnatal rat lung, lgl1 concentrates at the tips of budding secondary alveolar septa. We studied two models of impaired postnatal alveologenesis generated by exposure of newborn rats to 60% O2 for 2 wk or 95% O2 for 1 wk. A profound decrease of lgl1 expression with oxygen exposure was observed in both animal models. Animals exposed to 95% O2 for 1 wk recovered in air over a 3-wk period, associated with normalization of lgl1 levels. Changes in lung levels of alpha-actin (a marker of myofibroblast differentiation associated with alveologenesis) and the mesenchymal marker vimentin were significant but less marked. Our findings support a role for lgl1 in postnatal lung development. We speculate that deficiency of lgl1 contributes to the arrested alveolar partitioning observed in BPD and that recovery is associated with normalization of lgl1 levels.