Troubleshooting RNA Electrophoresis
Problems:
Problem 1 RNA bands are not visible
Possible cause Recommended solution Insufficient staining Use 2X RNA Loading Dye for both conventional RiboRuler™ RNA ladder and RNA sample preparation prior to electrophoresis. This solution includes ethidium bromide at a concentration sufficient to stain RNA on denaturing formaldehyde agarose gels. Ready-to-use RiboRuler™ RNA ladders are already premixed with 2X RNA Loading Dye.
If RNA fragments are separated on native agarose gels, additional staining with ethidium bromide (final concentration 0.5 µg/ml) is recommended. If RNA is separated on a denaturing glyoxal/DMSO agarose gel, stain the gel in ethidium bromide solution (final concentration 0.5 µg/ml) in 0.5 M ammonium acetate for 15 min after electrophoresis. Wash the gel in a fresh 0.5 M ammonium acetate solution for 15 min. If RNA is separated on a denaturing polyacrylamide gel with urea, soak the gel for about 15 minutes in 1X TBE to remove the urea prior to staining. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min. No staining If you are using home-made loading dye which does not contain ethidium bromide, add ethidium bromide to both agarose gel and electrophoresis buffer at a final concentration of 0.5 µg/ml.
Alternatively, stain the gel after electrophoresis with ethidium bromide (0.5 µg/ml ethidium bromide), or SYBR® Green II. Insufficient amount of ladder was loaded Follow the recommendations for loading for the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl/ per mm gel lane for ready-to-use ladders).
RNA degradation under UV light Minimize exposure to UV light as this may cause RNA degradation/fading. RNA diffusion from the gel Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion of smaller RNA fragments from the gel.
Avoid long term storage of the gel before taking a picture, as this may cause diffusion of RNA fragments and band fading. RNA has run out of the gel Perform electrophoresis until the bromophenol blue passes two thirds of the gel. In most denaturing agarose gel systems, bromophenol blue migrates slightly faster than 5S rRNA and xylene cyanol FF migrates slightly slower than 18S rRNA.
Problem 2 Smeared RNA bands
Possible cause Recommended solution RNA degradation by nucleases Any RNA, including RiboRuler™ RNA ladders, is extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly poured gels, diethyl pyrocarbonate (DEPC)-treated solutions and protective gloves is recommended. Improper storage or use of RNA ladders Store RiboRuler™ RNA ladders at -20°C for 6 months or at -70°C for 24 months. Thaw the ladders on ice. Gel too thick or too large a volume of RNA loaded Use thin (~0.5 cm) gels and avoid loading of large volumes in the gel lane. Improper electrophoresis conditions Ensure that there is enough electrophoresis buffer in the electrophoresis apparatus to ensure that the gel is immersed completely. Do not use an excessively high voltage for electrophoresis. Run the gels at 5 V/cm. To increase the band sharpness, use a lower voltage for several minutes at the beginning of electrophoresis. Too low of a voltage during the entire run may result in diffusion of bands during electrophoresis. Too much ladder loaded Follow the recommendations for loading for the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl/ per mm gel lane for ready-to-use ladders).
Problem 3 Atypical banding pattern
Possible cause Recommended solution Inefficient denaturation of the ladder All RiboRuler™ RNA ladders should be heated at 70°C for 10 min and chilled on ice before loading on the gel in order to completely denature the RNA. Sample RNA should be prepared the same way. Old formaldehyde used for preparation of the gel Older formaldehyde has an acidic pH what may cause extra RNA bands on the gel. Use only fresh formaldehyde for best results. Different loading conditions for the sample and the ladder RNA Both the ladder and the sample RNA should be prepared with the same DNA loading dye and loaded under the same conditions. After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human rRNA should be clearly visible under UV illumination. Fast-migrating bands composed of 5.8S RNA and 5S RNA may also be visible depending on the RNA purification procedure. The intensity of the 28S RNA should be approximately twice the intensity of the 18S RNA. The 28S human rRNA band migrates approximately with the 5000 b band, the 18S human rRNA band migrates approximately with the 1900 b band. Improper electrophoresis conditions Excessively long electrophoresis runs may result in migration of small RNA fragments out of the gel.
Very short electrophoresis may result in incompletely resolved bands.
Run gels at 5 V/cm until the bromophenol blue passes 2/3 of the gel.TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 b RNA and for denaturing polyacrylamide gel electrophoresis. The correct gel percentage is important for optimal separation of the ladder RNA; take into account the following: RiboRuler™ High Range RNA Ladder can be loaded on:
- native 0.8-1.5% agarose gel with TAE buffer
- denaturing formaldehyde 0.8-1.5% agarose gel with MOPS buffer
- denaturing glyoxal/DMSO 0.8-1.5% agarose gel with sodium phosphate buffer
RiboRuler™ Low Range RNA Ladder can be loaded on:
- native 1.7-2.5% agarose gel with TAE buffer
- denaturing formaldehyde 1.7-2.5% agarose gel with MOPS buffer
- denaturing glyoxal/DMSO 1.7-2.5% agarose gel with sodium phosphate buffer
- denaturing 4-8% polyacrylamide gel with TBE buffer
Problem 4 High background staining
Possible cause Recommended solution Excessively high ethidium bromide concentration or prolonged staining Use ethidium bromide at a final concentration of 0.5 µg/ml. Avoid prolonged staining of the gels. Insufficient gel destaining If the gel is extensively stained with ethidium bromide, additional destaining in water is needed to remove background staining. Wash glyoxal/DMSO agarose gels after staining in a fresh 0.5 M ammonium acetate solution for 15 min.
Problem 5 Uneven staining of the gel
Possible cause Recommended solution Improper gel staining conditions Ethidium bromide migrates in the opposite direction of the RNA during electrophoresis. Therefore, if ethidium bromide is only added to the agarose gel and not to the electrophoresis buffer, it may result in uneven RNA fragment staining. When 2X RNA Loading Dye is used for both conventional RiboRuler™ RNA ladders and RNA sample preparation prior to electrophoresis, additional staining is not required as the loading dye includes sufficient ethidium bromide to stain RNA on denaturing formaldehyde agarose gels. For native agarose gels, ethidium bromide (0.5 µg/ml) should be added to both the electrophoresis buffer and the agarose. This ensures an even distribution of ethidium bromide during electrophoresis so that the intensity of the bands upon exposure to UV light will be proportional to the quantity of RNA present. Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus or enough of the staining solution during the staining so that the gel is always immersed completely.
Problem 6 RNA remains in the gel lane
Possible cause Recommended solution Poorly-formed gel wells Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately.
Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample.Overloading of the gel Follow the recommendations for loading for the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl/ per mm gel lane for ready-to-use ladders). Contamination of the RNA sample Make sure that your sample RNA solution does not contain any precipitate.
Problem 7 Gel quantification data differs from spectrophotometrical data
Possible cause Recommended solution Contamination of the RNA sample Free NTPs and truncated transcripts remaining in the sample after in vitro transcription can interfere with spectrophotometrical measurements and lead to inaccurate quantification of sample RNA.
RiboRuler™ RNA ladders are produced from chromatography-purified RNA transcripts and are free of any NTPs and truncated transcripts. Therefore the gel quantification data is compatible with the spectrophotometrical measurements of RiboRuler™ RNA ladders.Incorrect RiboRuler™ RNA ladder band chosen for quantification of the sample Always compare the sample band with a similar size ladder band. Different loading conditions for the sample and the ladder RNA Both the sample RNA and ladder RNA should be loaded under the same conditions. Use the supplied 2X RNA Loading Dye both for sample RNA and ladder RNA. Loading equal volumes of sample RNA and ladder RNA is recommended. The required volume of sample RNA can be obtained by diluting with a mixture (1:1) of DEPC-treated Water and 2X RNA Loading Dye. Improper quantification method used If possible, quantify by video-densitometry measurements while subtracting the gel background as this method is more precise than a visual comparison of the bands. Uneven staining of the gel and high background staining Uneven staining of the gel and high background staining can also interfere with gel quantification results.
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Updated vasario 01, 2008 10:42
