Computational protocol: Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

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Protocol publication

[…] The jejunal mucosa was physically separated from seromuscular layers by scraping with a glass slide and was placed in RNAse free microtubes and immediately placed in liquid nitrogen. They were then stored at −80°C until use. Total RNA from jejunal tissue was extracted using the Qiagen RNeasy Minikit (Qiagen, Valencia, CA). Yield and quality of the extracts were determined by measuring absorbance at 260 and 280 nm (NanoDrop Technologies Thermo Fisher Scientific Wilmington, DE). The ratio of absorbance at 260∶280 was between 2.03 and 2.07. 1 µg of RNA was converted to cDNA using the iScript cDNA synthesis kit (Biorad) and pooled. The cycle conditions were 5 min at 25°C, cDNA synthesis at 42°C for 30 min, denaturation at 85°C for 5 min and held at 4°C. Primers were designed based on published sequences of the pig target genes either manually or using the NCBI online primer design tool (Primer-BLAST, http://www.ncbi.nlm.nih.gov/tools/primer-blast/), Primer3 input (version 0.4.0, frodo.wi.mit.edu/). The specificity of the primers was checked using the NCBI online Blast tool (Primer-BLAST, http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Quantitative RT-PCR was performed utilizing the iTaq Universal SYBR green Supermix (BioRad). Standard curves were generated using serial dilutions of pooled cDNA from all three normal pigs tested in triplicate. The StepOnePlus Real time PCR system (Applied Biosystems by Life Technologies, Carlsbad, CA) was used. Cycle parameters included polymerase activation and DNA denaturation at 95°C for 30 sec. Forty cycles of amplification were performed with a 15 sec denaturation at 95°C and annealing/extension and plate read for 60 sec at 60°C. The melting curve analysis was performed at 65°C–95°C at 0.5°C increments, 5 sec per step. Melting curves were checked to ensure consistent amplification of a single PCR product. Primer efficiency was calculated using the equation, Efficiency  = 10∧(−1/slope) –1. All primer efficiencies were greater than 92%. […]

Pipeline specifications

Software tools Primer-BLAST, Primer3
Application qPCR
Organisms Sus scrofa, Mus musculus, Homo sapiens
Chemicals Bromodeoxyuridine, Somatostatin