Separation of long RNA by agarose-formaldehyde gel electrophoresis

Abstract

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative "pK-matched" buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.

Keywords: Electrophoresis; Northern blotting; Pre-rRNA; RNA; rRNA biogenesis.

Fig. 1

(A, B) SYBR Gold stained gels of RNA separated with the standard and optimized electrophoresis procedures as detailed in the text. We used a 7.5×10 cm mini-gel horizontal system (C.B.S. Scientific) to run a 1% agarose gel at 100 V (6 V/cm) for 2 h; bromophenol blue migrated ~7 cm in all cases. Marker lanes (1–2) contained commercial RNA ladders (Thermo Fisher Scientific #SM1833 and #SM1823). Lanes 3–5, total RNA extracted from Saccharomyces cerevisiae cells. Lanes 6–7, total RNA from mouse 3T3 fibroblasts. Equal amounts of RNAs were loaded on all gels. rRNA bands look less intense in the standard protocol because of a lower efficiency of SYBR Gold staining in the MOPS buffer, as compared with HT and TT buffers; RNA ladders were premixed with 0.0125% ethidium bromide. (C, D) Northern hybridization of mouse rRNA precursors separated on gels as described in panels A and B. RNA was hybridized [9] with probe ITS1-1c [10] and visualized using a Typhoon 8600 Phosphorimager (GE Healthcare Life Sciences). Positions of 45S (12.8 kb), 41S (8.8 kb), 36S (6.9 kb), 34S (6.3 kb) and 20S (2.9 kb) pre-rRNAs are indicated.