Millennium™ RNA Markers

(1) RNA was not completely denatured. Electrophorese RNA under denaturing conditions. Use urea, formamide or formaldehyde gels, or glyoxal-treated RNA.

(2) For glyoxal-treated RNA, a buffer gradient formed during electrophoresis. Recirculate buffer during electrophoresis to prevent gradient formation.

Extra bands appear in RNA Ladders for a few reasons:

(1) RNA was not completely denatured. Electrophorese RNA under denaturing conditions. Use urea, formamide or formaldehyde gels, or glyoxal-treated RNA.

(2) Extra bands may be a result of using formaldehyde that is not fresh; the pH becomes acidic in older formaldehyde. In our hands, when fresh formaldehyde with neutral pH was used, the extra bands disappeared.

(3) Alternatively, if the extra bands appear after hybridization, it could be that the gel purified probe contains contaminating vector DNA (pUC or pBR) that hybridizes to RNA Ladder template DNA.

Missing RNA bands may be due to:

(1) A small amount of RNA diffused out of gel during extended destaining. Minimize destaining time. Destaining for 2 hours is sufficient for most applications.

(2) RNA bands of similar molecular size were not resolved. Use the correct gel type and denaturing conditions.

RNA was exposed to UV light for extended periods of time. Minimize exposure to UV light. Stain and destain gels in the dark and photograph the gel immediately.

Many factors could affect the intensity of the bands as summarized below.

(1) Insufficient RNA was loaded on the gel. Increase the amount of RNA loaded.

(2) RNA was degraded. Avoid nuclease contamination of the RNA standards. Store RNA at -70 degrees C in formamide. Deionize formamide and glyoxal, store aliquots at -20 degrees C.

(3) RNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel.

(4) For ethidium bromide-stained RNA, improper UV light source was used. Use short-wavelength (254 nm) UV light. For ethidium bromide-stained RNA, improper staining and destaining conditions were used. Stain in the dark with 5 mg/mL ethidium bromide. For thin (3.2 mm) formaldehyde gels, stain 5 min and destain 1 h. For thicker gels, stain 30 min and destain 2 hr. To reduce background staining, use 0.66 M formaldehyde instead of 2.2 M formaldehyde in gels. For glyoxal RNA gels, stain and destain in 0.5 M ammonium acetate to help reduce background staining.

(5) For radiolabeled RNA, an improper labeling method was used.