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Frequently Asked Questions: RiboRuler™ RNA Ladders (2-3)
1. What is the composition and what are the benefits of the 2X RNA Loading Dye supplied with RiboRuler™ RNA ladders?2X RNA Loading Dye (supplied with every Fermentas RiboRuler™ RNA ladder) contains:
95% formamide
0.025% SDS
0.025% bromophenol blue
0.025% xylene cyanol FF
0.025% ethidium bromide
0.5 mM EDTAIt has several benefits:
- It contains high purity formamide, a denaturing agent - in most conditions, this allows RNA molecules to be separated according to their true size even during non-denaturing electrophoresis. In addition, formamide and SDS stabilize RNA, reducing the possibility of degradation.
- The electrophoresis tracking dyes bromophenol blue and xylene cyanol FF help to monitor the progress of electrophoresis. In most denaturing agarose gel systems, bromophenol blue migrates slightly faster than 5S rRNA and xylene cyanol FF migrates slightly slower than 18S rRNA.
- EDTA binds divalent metal ions, inhibits metal dependent enzymatic reactions and protects RNA from degradation by metal dependent nucleases.
- The presence of the intercalating dye ethidium bromide in the loading buffer has several advantages over other RNA staining approaches:
- only the RNA bands are stained, resulting in less background staining and higher sensitivity compared to gel/buffer staining;
- gels do not require time-consuming destaining/staining procedures, which may cause RNA diffusion. As a result RNA bands are sharper as well as more intense;
- requires less ethidium bromide than other straining methods. This prevents from RNA mobility shifts that are sometimes caused by a high concentration of ethidium bromide;
- no interference with Northern blots and hybridizations has been observed.
2. Are there any electrophoresis conditions for which additional staining of the gel is required?
- For standard denaturing formaldehyde agarose gels, 2X RNA Loading Dye is optimized and includes sufficient ethidium bromide.
- For denaturing glyoxal/DMSO agarose gels, stain the gel in a 0.5μg/ml ethidium bromide/0.5 M ammonium acetate solution with gentle shaking for 15 min. Wash the gel in fresh 0.5 M ammonium acetate solution with gentle shaking for 15 min prior to visualization of the fragments under UV illumination.
- For denaturing polyacrylamide gels with urea, soak the gel in the 1xTBE for 15 minutes prior to staining to remove the urea. Stain the gel in a 0.5μg/ml ethidium bromide/ 1X TBE solution for 15 min if necessary.
- For native agarose gels, additional staining with ethidium bromide during gel electrophoresis is recommended. Ethidium bromide should be added to both the electrophoresis buffer and the agarose gel at a final concentration of 0.5 μg/ml. This ensures an even distribution of ethidium bromide during electrophoresis so that the band intensity upon exposure to UV light will be proportional to the quantity of RNA present in the bands. Alternatively, the gel can be stained after the electrophoresis by immersing it for 10-15 minutes in water containing 0.5 µg/ml ethidium bromide.
Note
Ethidium bromide migrates in the opposite direction to RNA during electrophoresis. Therefore, if ethidium bromide is added only to the agarose gel and not the electrophoresis buffer, uneven staining of RNA fragments will result.
3. Is the 2X RNA Loading Dye suitable for electrophoresis of native RNA?The 2X RNA Loading Dye is not suitable for analysis of native RNA as it contains the denaturing agents, formamide, SDS and ethidium bromide.
To analyze native RNA molecules, loading dye without denaturing agents should be used. The ladder and the sample RNA should be prepared using the same DNA loading dye.4. Can a home-made loading dye solution be used for electrophoresis of RiboRuler™ RNA ladders?We guarantee the quality of our RiboRuler™ RNA ladders when they are used with the supplied 2X RNA Loading Dye in the recommended conditions. Use of the supplied loading dye minimizes the risk of band degradation and gives the most accurate results.
If there is a strong need to use a home-made loading dye solution, please follow below recommendations:
- Load both the sample and the ladder RNA using the same loading dye solution. For the most accurate results, the same volume of both the ladder and sample should be loaded.
- Do not exceed 0.0125% final ethidium bromide concentration in the loading dye, or the migration of the RNA will be altered due to changes in its charge.
III. RNA quantification on gels
1. Can RiboRuler™ RNA ladders be used for RNA quantification on gels?
- RiboRuler™ RNA ladders are ideal for RNA quantification on gels. Our exceptionally pure ladders are produced from chromatography-purified RNA transcripts. They are free of any NTPs and truncated transcripts that could interfere with spectrophotometric measurements and lead to inaccurate RNA quantification.
- The RNA concentration of each ladder band is indicated next to the image of each ladder provided in the Certificate of Analysis of the product.
2. How can the quantification accuracy be maximized?
- Use the supplied 2X RNA Loading Dye both for sample RNA and ladder RNA.
- Load equal volumes of the sample RNA and the RiboRuler™ RNA ladder.
- Adjust the RNA sample volume by diluting with equal volumes of DEPC-treated Water and 2X RNA Loading Dye.
- 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.
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Updated vasario 01, 2008 09:14
