C. C. Culbreath1, B. Halloran2, D. K. Crossman3, N. Ambalavanan2, T. Jilling2, C. A. Martin1 1University Of Alabama At Birmingham,Pediatric Surgery/Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Neonatology/Pediatrics,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Bioinformatics/Genomic Science,Birmingham, Alabama, USA
Introduction: Necrotizing enterocolitis (NEC) continues to be the leading gastrointestinal cause of demise in premature infants. Lack of representative animal models is a challenge in clarifying the pathogenesis of NEC. In depth molecular analysis of human tissue is hence required for understanding the mechanisms involved in NEC development.
Methods: Six premature infants that had a surgical resection for NEC and 6 age-matched ileal atresia controls were identified from the medical records. RNA was isolated from formalin fixed paraffin embedded tissue sections using the Rneasy FFPE kit from Qiagen. RNA quality was verified using an Agilent Bioanalyzer and by performing reverse transcription real time PCR analysis of housekeeping genes (GAPDH and 18S). Only RNA that exhibited good quality with both methods was used for microarray analysis. Gene expression analysis by microarray was then performed with the Illumina HT-12_V4 chip. A subsequent Ingenuity Pathway Analysis (IPA) was then done to identify top canonical pathways and regulators involved. All studies were done in accordance after approval by the University of Alabama at Birmingham Institutional Review Board.
Results:Greater than 47,000 genes were screened. A total of 47 genes were found to have a fold change > 2, and 37 genes had a fold change > 2 and p < 0.05. Pathway analysis determined top canonical pathways (Table 1), top upstream regulators, top diseases and biofunctions, top organ toxicity functions, and top regulator effector networks. The top upstream regulators were STAT3, Prolactin (PRL), IL1b, STAT1, and IFNg.
Conclusion:Paraffin sections are a reliable source of quality RNA for microarray analysis. Identification of relevant pathways and signal transducers by pathway analysis are being validated by protein expression to guide future studies in understanding mechanistic pathways resulting in necrotizing enterocolitis.
