Computational protocol: Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

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Protocol publication

[…] Total RNA and cDNA was obtained from fetal lungs using TRIzol reagent (Invitrogen, Carlsbad, CA) and M-MLV Reverse Transcriptase (Promega, Madison WI), respectively, as per the manufacturer’s instructions. The integrity of 28S and 18S rRNA from total RNA was always assessed on agarose gels prior to further analysis. qPCR analysis was performed in triplicate for each biological replicate using at least two housekeeping genes per analysis: 18S rRNA and Rps29. Other qPCR primers were designed to include at least one oligo overlapping an exon-exon boundary, using the web-based software Primer3. Primer efficiency was then calculated using a standard curve and fetal lung cDNA template. Sequences for these primers can be found in a previous publication. Cycling was performed on a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Richmond, CA). Relative differential expression was then determined using CFX Manager™ software (Bio-Rad). […]

Pipeline specifications

Software tools Primer3, CFX Manager
Application qPCR
Organisms Homo sapiens, Mus musculus