Computational protocol: Density of human bone marrow stromal cells regulates commitment to vascular lineages

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Protocol publication

[…] Total RNA was isolated from cultured cells using Trizol reagent. Total RNA (1 μg) was reverse-transcribed and subjected to polymerase chain reaction (PCR). Quantitative PCR was performed using a SYBR green quantitative PCR core kit (Eurogentec), with each sample run in triplicate. Data were normalized to GAPDH, and relative expression was calculated according to the 2-[delta]CT formula. Oligonucleotide primers for PCR were designed using Primer3 software. Each primer pair was designed using the same parameters (70- to 100-bp product size and Tm of 60 °C). PCR were repeated using a second primer set (sequences not shown). Reaction products were resolved using 2.5% ultrapure agarose (Invitrogen) gel electrophoresis run at 100 V for 2 h in 1X TAE (Tris base, acetic acid, EDTA) buffer containing 2.5 μl Gel Red (Biotium), and visualised using a transilluminator. [...] In all quantitation experiments, results were expressed as the mean ± standard deviation. Statistical differences between sets of data were determined using a paired t test on SigmaPlot 8.0 software, with P < 0.05 considered significant. […]

Pipeline specifications

Software tools Primer3, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Homo sapiens