Isolation of Translationally Active mRNA

This protocol was adapted as previously described²¹ (link) and modified accordingly. In brief, we prepared the cytoplasmic extracts by harvesting proband and control cells in ice-cold PBS containing 100 μg/mL cycloheximide (Sigma). Cells were counted, and 100,000 cells were incubated with 800 μL of RPMI medium containing 10% fetal bovine serum and 100 μg/mL cycloheximide (Sigma) for 5 min at 37°C. After incubation, 200 μL of N-hydroxysuccimide ester (DSP; 1 mM; Pierce) was introduced as a cross-linking reagent and incubated for 5 min at 37°C followed by quenching with 1 M Tris–HCl (pH 7.4). The cells were washed twice by centrifugation at 1,000 r.p.m. for 3 min and rinsed with ice-cold PBS containing 100 μg/mL cycloheximide (Sigma). The final pellets were swollen for 20 min in 500 μL of low-salt buffer (LSB) (20 mM HEPES [pH 7.4], 100 mM KCl, and 2 mM MgCl₂) containing 1 mM dithiothreitol and lysed by the addition of 500 μL lysis buffer (1× LSB containing 1.2% Triton X-100) (Sigma) followed by brief vortexing. One-tenth (70 μL) of the above lysate was transferred to the Ig-coated beads, and incubation was carried out for 2 h at 4°C. After incubation with the HSP70/HSP73 antibody-conjugated magnetic beads, the polysome complexes containing translationally active mRNA transcripts were isolated and eluted from beads with the Array Pure Nanoscale RNA Purification Kit (Epicentre).