Fla 5100 scanner

Total RNA was extracted from cell lysates by GTC-Phenol extraction. 10 μg total RNA was separated on an 8% polyacrylamide TBE-Urea gel and transferred to a nylon membrane (HyBond N+, GEHealthcare, RPN1210B) by electroblotting for 4 hr at 50 V. Membranes were pre-hybridised in 10 ml of UltraHyb Oligo Hyb (Thermo Scientific, AM8663) for 1 hr and probed with ³²P-labeled DNA oligo at 42°C for 12–18 hr in a hybridization oven. The sequences of the probes used for Northern blot detection are detailed in Supplementary file 10. Membranes were washed twice with 2xSSC + 0.5% SDS solution for 10 min and visualized using a Phosphor imaging screen and FujiFilm FLA-5100 Scanner (IP-S mode). For detection of highly abundant species (5S rRNA) autoradiography was used for exposure.

The 2D protein separation was carried out as described earlier30 (link). The 2-DE gels were stained with Flamingo fluorescent gel stain (Bio-Rad, Hercules, CA, USA) following the manufacturer instructions. After staining, gels were scanned at 50 μm resolution on a Fuji FLA-5100 scanner. The digitalized images were analyzed using Delta 2D 3.4 (Decodon, Brunswick, Germany). For protein visualization, the 2-DE gels were additionally stained overnight with colloidal Coomassie blue, Roti-Blue (Roth, Karlsruhe, Germany).

One microgram total RNA was reverse-transcribed using SuperScript III reverse transcriptase (Thermo Scientific, 18,080,051) using ³²P -radiolabelled oligonucleotides as primers (Supplementary Table 4). Primers were added to the RNA and annealing was performed by heating the samples at 85°C for 3 min and then snap chilling them on ice. The RT was performed for 1 h at 45°C, followed by Exonuclease I and RNaseIf (NEB M0293L and M0243S) (0.5 μl each) treatment for 30 minutes at 37°C. Reactions were stopped by mixing with an equal volume of 2XRNA loading dye (NEB, B0363S), 2 minute incubation at 95°C and snap chilled. The sequencing ladders were prepared with Sequenase v2.0 (Thermo Scientific, 70775Y200UN) according to specified instructions. Samples were resolved on 6% PAA/8 M TBE-urea gels and visualized using the FujiFilm FLA5100 scanner.

Electrophoretic mobility shift assays (EMSA) were performed with internally labeled pre-miRNA transcript and proteins produced in Escherichia coli. Gel-purified probes (50 × 10³ c.p.m. [counts per minute], ∼20 pmol) were incubated in 15-µL reaction mixtures containing the indicated amounts of proteins in Roeder D buffer (100 mM KCl, 20% [v/v] glycerol, 0.2 mM EDTA, 100 mM Tris at pH = 8.0, 0.5 mM DTT, 0.2 mM PMSF) supplemented with 0.5 mM ATP, 20 mM creatine phosphate, and 3.2 mM MgCl₂. Reactions were incubated at 4°C for 1 h followed by electrophoresis on a 6% (w/v) nondenaturing gel. The signal was registered with radiographic film or was exposed to a phosphoimaging screen and scanned on a FLA-5100 scanner (Fujifilm).

Validation of the 2-DE data was carried out using western blot (WB) analysis. To assure the reproducibility of the WB analysis, at least three experimental replicates from each patient’s serum were performed. Proteins (40 μg) were separated by SDS-PAGE and transferred to Hybond® ECL nitrocellulose membrane (GE Healthcare, Freiburg, Germany). Immunodetection was performed as described previously [21 (link)]. Briefly, membranes were blocked in 5% milk for 2 hours at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibody including polyclonal rabbit anti-human haptoglobin (Hp; 1:1,000), rabbit anti-human apolipoprotein (Apo) C-III antibody (Genway, San Diego, CA, USA), rabbit anti-human ApoA-II antibody (Genway) or a rabbit anti-human vitamin D-binding protein antibody (Abcam, Cambridge, UK).
After washing three times in Tris-buffered saline–Tween buffer, nitrocellulose membranes were incubated with the corresponding secondary antibody (Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG, 1:2,000; Molecular Probes, Darmstadt, Germany). Air-dried blot membranes were then scanned at 50 μm resolution on a Fuji FLA-5100 scanner with single laser-emitting excitation light at 635 nm.