Fig. 4.

Optimization of conditions for transcript-specific affinity capture. (a) Northern blot analysis of GFP mRNA (target RNA) captured from total purified RNA under varying hybridization conditions. Variables shown include bead volume, hybridization volume, salt concentration, and the addition of formamide to hybridization reactions. Percent recovered in eluate relative to input indicated below. ND not detected. Note, a number of additional optimization conditions were tested (not shown) and all conditions leading to increased capture of the target RNA were incorporated into the final protocol. (b) Northern blot analysis of GFP mRNA (target RNA) captured from total purified RNA under varying hybridization times and temperatures. RT room temperature, O/N overnight, NC not calculated