RIPK2 protein, human

We evaluated the manner in which the local maxima of the probability distribution approximated the nearest hydration sites using the mean absolute positional deviation (MAD) and root-mean-square deviation (RMSD) scores, defined as follows: $$MAD=\frac{1}{N}\sum _{i=1}^N\left|{\mathbf{r}}_i^c-{\mathbf{r}}_i^p\right|,RMSD=\sqrt{\sum _{i=1}^N{\left({\mathbf{r}}_i^c-{\mathbf{r}}_i^p\right)}^2/N},$$ where ${\mathbf{r}}_i^c$ and ${\mathbf{r}}_i^p$ are the positions of an experimentally identified hydration site and the local maximum of probability distribution near the site, respectively. $N$ is the number of hydration sites targeted in the evaluation.

Six differentially expressed genes were selected for validation using quantitative real-time PCR (qPCR). The 500 ng of sample RNA and reference RNA (Universal Human Reference RNA, Invitrogen) was reversely transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). In the less concentrated samples, the initial concentration of 250 ng was processed to reverse transcription (RT). No Enzyme Control (NEC) was also included in each series of transcription. The temperature steps of RT include incubation at 25°C for 10 min, RT at 37°C for 120 min, and enzyme inactivation at 85°C for 5 min. Samples were stored at −20°C until further use.
The initial step of relative quantification (RQ) includes 10-fold serial dilutions of reference RNA from 25 ng to 2.5pg. The six target assays, namely, receptor interacting serine/threonine kinase 2 (Applied Biosystems, RIPK2, Hs01572686_m1, FAM-MGB), interleukin 6 receptor (IL6R, Hs01075664_m1, FAM-MGB), TNF superfamily member 10 (TNFSF10, Hs00921974_m1, VIC-MGB), MHC class I polypeptide-related sequence B (MICB, Hs00792952_m1, FAM-MGB), C-C motif chemokine receptor 4 (CCR4, Hs01396342_m1, FAM-MGB), and B-cell linker (BLNK, HS00179459_m1, VIC-MGB), and two housekeeping genes, actin beta (ACTB, HS99999903_m1, VIC-MGB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1, VIC-MGB), were tested. Each duplex reaction was run with TaqMan™ Fast Advanced Master Mix (Applied Biosystems) in the total volume of 20 μl with thermal cycling conditions, as incubation at 50°C for 2 min, polymerase activation at 95°C for 2 min, and 40 cycles with denaturation at 95°C for 3 s and annealing/extension at 60°C for 30 s in the instrument 7500 Fast Real-Time PCR System (Applied Biosystems). Standard curves were created for each tested assay from dilution series in the 7500 instrument software, and pairs of assays labeled with FAM and VIC have been chosen for duplex reactions (IL6R+BLNK, MICB+TNFSF10, RIPK2+GAPDH, and CCR4+ACTB). Data are not present in the study.
For validation of RNA sequencing by RQ, 91 tumors and 71 adjacent tissues were selected. Samples were analyzed in duplicates for each duplex reaction, and all pipetting steps were performed on BRAVO Liquid Handling Station (Agilent) to minimalize subjective pipette handling bias. The results were analyzed in the instrument software with unique threshold setting and cycle threshold (Ct value)calculation.