Wizard plus minipreps dna purification system

Urine samples of kidney cancer were recruited from the Department of Surgery of the Prince of Wales Hospital, Hong Kong. Healthy samples were recruited from the Department of Chemical Pathology of the Prince of Wales Hospital, Hong Kong. All subjects involved in this study gave written informed consent, and the study was approved by The Joint Chinese University of Hong Kong–Hospital Authority New Territories East Cluster Clinical Research Ethics Committee, under the Declaration of Helsinki. Collection and storage of fresh urine were followed as previously described^{17 (link)}. Urinary DNA was extracted from 10 mL–30 mL cell-free urine component with the Wizard Plus Minipreps DNA Purification System; (Promega). 15 mL of 6 mol/L guanidine thiocyanate (Sigma–Aldrich) and 1 mL of resin (Wizard Plus Minipreps DNA Purification System; Promega) were added in each 10 mL of the processed urine. The mixture was incubated and shaken gently at room temperature for 2 h. Then, the DNA was purified and eluted into 100 μL RNase-free water using the minicolumns and according to the manufacturer’s instruction of the purification system. The quantity of urinary DNA obtained ranged from ~10 ng to ~100 ng according to Qubit assays.

The pvsoap gene was amplified using primers pvSOAP-F and pvSOAP-R (Supplementary Table S1). For PCR reactions, 100 ng of total DNA, 0.25 mM of each primer, 0.25 mM dNTPs, 1X buffer (100 mM KCl, 20 mM Tris–HCl pH 7.5), and 2.4 mM MgSO₄ for 2 U of High Fidelity PlatinumR Taq DNA polymerase (Invitrogen Carlsbad CA, USA) were used. Reaction conditions were as follows: 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s; these were held at 72°C for 5 min. DNA fragments were cloned using a kit (TOPO TA Cloning, Invitrogen Carlsbad CA, USA) according to manufacturer's instructions. Plasmids were extracted using the kit Wizard plus Minipreps DNA Purification System (Promega_Madison WI, USA).

T helper cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS), lysed with RLT buffer and RNA was extracted (RNAeasy mini kit Qiagen, Gaithersburg, MD) per manufacturer’s instructions. cDNA was synthesized (First-Strand cDNA synthesis Origene, Rockville, MD) per manufacturer’s instructions. Quantitative PCR (qPCR) was performed on a 7300 Real Time PCR System (Applied BioSystems, Foster City, CA). Primers were designed using Integrated DNA technology website (http://www.idtdna.com/Primerquest/Home/Index) with sequences from Genebank accession numbers (Table 1). Changes in gene expression were calculated by the ΔΔCT method[47 (link)] and depicted as fold change in gene expression compared to control.
To confirm the specificity of our primers, qPCR products were run on a 2% agarose gel and product size as well as the presence of non-specific products were determined. IL17A and RORc qPCR products were further cloned into 2.1 PCR product vector per manufacturer’s instructions (TOPO-TA Cloning Kit, Life technologies). Plasmids were then transfected into OneShot^® cells and positive colonies selected and expanded overnight prior to plasmid isolation (Wizard^® Plus Minipreps DNA Purification System, Promega, Madison, WI) and sequencing by an automated sequencer (Nucleics, Davis, CA).