Data were entered into OpenClinica version beta 3.1.2 clinical trial software (Isovera Inc, Boston, MA, USA) for clinical data management, using the double-entry method by at least two independent researchers for completeness.
Apoptosis for B-CLL and healthy cells was determined by FACS analysis (FACS Canto II, BD BioSciences, Franklin Lakes, NJ, USA) using a double staining with Annexin V-FLUOS (Roche, Basel, Switzlerland) and propidium iodide (PI; Sigma Aldrich, St . Louis MO, USA) as reported before.
Remember when you used the double lift?
Quantitative PCR was performed with the real-time PCR system, iQ™ 5 (Bio-Rad, Hercules, CA, USA), using SYBR Green I double strand DNA binding dye.
For double staining, CTA 157-2 was linked to NHS-Rhodamine (Pierce, Rockford, USA) using protocols provided by the manufacturers.
cDNA pools were subjected to real-time kinetic PCR on a Mx-4000 Multiplex Quantitative PCR (Stratagene, La Jolla, California, USA) using SYBR Green I as a double-strand DNA-specific binding dye to quantify mRNA expression for IL-12, IFN-γ, IFN-α, IFN-β, TNFα, IL-4, IL-5, IL-18, IL-23, IL-27, RRV structural protein VP6, and the nonstructural protein NSP3.
After separation, proteins were transferred to a PVDF membrane (Immun-BlotTM, Bio-Rad CA, USA) using an electrophoresis tank filled with a double concentrated running buffer without SDS supplemented with 10%% (v/v) methanol for 45 min at 100 V constant voltage.
The eluted RNA was treated with DNase I (DNA-free, Ambion, Austin, TX, USA), and double stranded DNA was generated using the Superscript double-stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA) primed with random hexamers.
HeLa cells (1.8×107 cells) were transfected with double-stranded duplex with Hs_HRMT1L2_7 HP Validated siRNA (Qiagen, Valencia, CA, USA) or luciferase (Dharmacon, Lafayette, CO, USA) using Lipofectamine reagent according to the manufacturer's recommendations (Invitrogen).
DNA concentration was quantified using the PicoGreen double strand DNA Quantification Kit (Molecular Probes Inc., USA).
