Pamm 026

Real Time PCR array reactions were performed as previously described (25 (link), 47 (link)). In short, extraction of total RNA from the remaining alveolus was performed with the RNeasy Plus kit (Qiagen Inc, Valencia, CA) according to the manufacturer’s instructions. The integrity of the RNA samples was verified by analyzing 1 µl of the total RNA in a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions, and the complementary DNA (cDNA) was synthesized using 1 mg of RNA through a reverse transcription reaction QuantiTect Rev Transcription kit (Qiagen Inc, Valencia, CA). Real Time PCR array was performed in a Viia7 instrument (Thermo Fisher Scientific, Carlsbad, CA) using a custom panel containing targets “Wound Healing” (PAMM-121), “Inflammatory cytokines and receptors” (PAMM-011), and “Osteogenesis” (PAMM-026) (SA Biosciences, Frederick, MD) for gene expression profiling. Real Time PCR array data were analyzed by the RT2 profiler PCR Array Data Analysis online software (SA Biosciences, Frederick, MD) for normalizing the initial geometric mean of three constitutive genes (GAPDH, ACTB, Hprt1) and subsequently normalized by the control group, and expressed as fold change relative to the control group.

Real-Time PCR array reactions were performed as previously described.^{4 (link)}
The extraction of total RNA from the remaining alveolus was performed with the RNeasy FFPE kit (Qiagen Inc., Valencia, CA) according to the manufacturers’ instructions. The integrity of the RNA samples was verified by analyzing 1mg of total RNA in a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturers’ instructions, and the complementary DNA was synthesized using 3μg of RNA by a reverse transcription reaction (Superscript III, Invitrogen Corporation, Carlsbad, CA). Real-Time PCR array was performed in a ViiA7 instrument (LifeTechnologies, Carlsbad, CA) using a custom panel containing the targets “Wound Healing” (PAMM-121), “Inflammatory cytokines and receptors” (PAMM-011), and “Osteogenesis” (PAMM-026) (SABiosciences, Frederick, MD), as well as for NOS isoforms (iNOS, nNOS, and eNOS), used for gene expression profiling. Real-Time PCR array data was analyzed by the RT2 profiler PCR Array Data Analysis online software (SABiosciences, Frederick, MD) to normalize the initial geometric mean of three constitutive genes (GAPDH, ACTB, Hprt1) and then normalized by the control group (comprising an additional independent group of mice, not subjected to any surgical procedure), and expressed as fold change regarding the control group, as previously described.^{4 (link)}