Computational protocol: The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration

Similar protocols

Protocol publication

[…] RT-PCR was performed using the GoTaq Green master mix (Promega, Madison, WI, USA). In each reaction, GoTaq Green master mix, forward primer (10 pmol), reverse primer (10 pmol) and nuclease-free water were added to cDNA in a 200 µl PCR tube. The primers used in this study are detailed in . Primer3 software was used to identify primer binding sites and predicted product size. The specific cycling conditions were 94°C for 5 min followed by 32–34 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 40 sec. Following this, a final extension was undertaken at 72°C for 10 min. Following PCR, samples were size separated using gel electrophoresis, stained in SYBR Safe (Life Technologies, Paisley, UK) and visualised under blue light using a Syngene U:Genius 3 system (Geneflow, Lichfield, UK). [...] The Ampliflor UniPrimer Universal system (Intergen Co., Purchase, NY, USA) was used to quantify relative transcript copy numbers in each cDNA sample as previously described (). Sample cDNA was combined with 2X iQ supermix (Bio-Rad Laboratories), forward primer (10 pmol), reverse primer containing the z sequence (1 pmol) and a UniPrimer probe (10 pmol). The specific primers are outlined in , and the cycling conditions were initial denaturing at 94°C for 5 min followed by 60 cycles of 94°C for 10 sec, 55°C for 15 sec and 72°C for 20 sec. The incorporation and unfolding of the UniPrimer probe, due to its similarity to the z sequence, separates a fluorophore from a quencher moiety and allows detection of the fluorescent signal which is indicative of DNA amplification. Quantification of this signal against a standard set of samples allows the quantification of relative transcript copy numbers. Sample quantity was further normalised against the GAPDH housekeeping gene levels present in the samples. [...] Cells were grown to confluence in a 24-well plate. Following this, the confluent monolayer was scratched using a 200 µl pipette tip to generate an artificial wound. Subsequently, the media was replaced with either normal media or media containing 100 ng/ml rhIL-15 and the wound tracked over a 120 min time period under an inverted microscope (Leica DMIL LED) on a heated plate at 37°C (both from Leica Microsystems GmbH, Wetzlar, Germany). Images were captured every 30 min using a digital camera (Leica DFC3000G; Leica Microsystems GmbH) and the distance between the wound fronts were calculated using ImageJ software. The distance migrated was calculated in comparison to the 0 h wound. [...] The SigmaPlot 11 statistical package was used for statistical analysis. Two-sample, two-tailed t-tests, Mann-Whitney U test and one or two way ANOVA analysis were undertaken depending on data normality. Experimental repeats were undertaken for all assays and p<0.05 was considered to indicate a statistically significant difference. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Neoplasms
Chemicals Calcium