Bleach gel: a simple agarose gel for analyzing RNA quality

Abstract

RNA-based applications requiring high-quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the 'bleach gel' is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.

Figure 1. Protective effect of bleach on RNA quality in RNase-containing gels

Standard 1% TAE agarose gels were made containing 20 ng/ml of RNase A along with various concentrations of household bleach (0% to 5.0% v/v Clorox®). Each gel was loaded with 10 μL 1× DNA Loading Buffer containing 1 μg of total RNA isolated from 4T1.2 mouse mammary carcinoma cells and run for ∼35 minutes at a constant 100 V. The presence of 28S, 18S, and 5.8S/5S ribosomal RNA (rRNA) bands are absent in the gel containing 0% bleach. Increasing rRNA band integrity occurs with bleach concentrations of 0.1 to 0.5% (a 0.5% bleach has 250 μl bleach/50 ml gel). The 28S, 18S, and 5.8S/5S rRNA bands are intact in gels with bleach concentrations of 1.0 to 5.0% (indicated by arrows). An increase in bleach concentration also results in a linear increase in amperage. These results suggest that a 1% TAE agarose ‘bleach gel’ is best run at a concentration of 0.5% to 1% v/v bleach.