Bioinformatic identification of novel early stress response genes in rodent models of lung injury

Abstract

Acute lung injury is a complex illness with a high mortality rate (>30%) and often requires the use of mechanical ventilatory support for respiratory failure. Mechanical ventilation can lead to clinical deterioration due to augmented lung injury in certain patients, suggesting the potential existence of genetic susceptibility to mechanical stretch (6, 48), the nature of which remains unclear. To identify genes affected by ventilator-induced lung injury (VILI), we utilized a bioinformatic-intense candidate gene approach and examined gene expression profiles from rodent VILI models (mouse and rat) using the oligonucleotide microarray platform. To increase statistical power of gene expression analysis, 2,769 mouse/rat orthologous genes identified on RG_U34A and MG_U74Av2 arrays were simultaneously analyzed by significance analysis of microarrays (SAM). This combined ortholog/SAM approach identified 41 up- and 7 downregulated VILI-related candidate genes, results validated by comparable expression levels obtained by either real-time or relative RT-PCR for 15 randomly selected genes. K-mean clustering of 48 VILI-related genes clustered several well-known VILI-associated genes (IL-6, plasminogen activator inhibitor type 1, CCL-2, cyclooxygenase-2) with a number of stress-related genes (Myc, Cyr61, Socs3). The only unannotated member of this cluster (n = 14) was RIKEN_1300002F13 EST, an ortholog of the stress-related Gene33/Mig-6 gene. The further evaluation of this candidate strongly suggested its involvement in development of VILI. We speculate that the ortholog-SAM approach is a useful, time- and resource-efficient tool for identification of candidate genes in a variety of complex disease models such as VILI.

Ischemic acute kidney injury induces a distant organ functional and genomic response distinguishable from bilateral nephrectomy

“…Quantitative real-time RT-PCR validation of selected candidate genes was conducted as previously described (23,24). Briefly, transcript levels of selected candidate genes in control and kidney IRI-treated lung tissues were measured (n ϭ 3/condition) in 96-well microtiter plates with an ABI Prism 7700 Sequence Detector Systems (PerkinElmer/Applied Biosystems).…”

“…Experimental parameters were 48°C for 30 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expression was calculated using the 2 Ϫ⌬⌬Ct method, which generates fold-change values for mRNA transcript levels expressed in IRItreated lung samples relative to sham-operated samples as described previously (24).…”

Exon-based mapping of microarray probes: Recovering differential gene expression signal in underpowered hypoxia experiment

“…were measured (n=3 per condition) as previously described [8]. Briefly, the 96-well microtiter plate setting of an ABI Prism 7700 Sequence Detector Systems (Perkin-Elmer/Applied Biosystems) was employed.…”

“…Our lab and the others have demonstrated that expression analysis of underpowered array experiments complemented with compatible expression information from similar experimental settings produced biologically feasible results [6][7][8]. In current studies, we further utilized complementary information obtained from multiple probe-sets that target the same exon or UTR .…”

Biomarkers of Acute Lung Lesion

“…Вероятно, расшифровка данных механизмов позволит определить процесс инициации ОПЛ и системных эффектов данного синдрома. Новые технологии должны определить роль каждого регуляторного белка в изолирован ных клетках или в ткани организма, развитие посттрансляционных изменений и межмолеку лярные взаимоотношения белков, определяющих реализацию фенотипа клетки [59,60].…”