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RNA Gels

Copyright 2012 - Molecular Station

History of RNA Gel Electrophoresis

RNA Gels have been used for decades to separate out and qualitatively assess the quality of RNA isolated from samples. If you are not sure what RNA is checkout:

AUDIO Molecular Biology Audio Listen to a detailed RNA Explanation! and see our RNA encyclopedia article.

RNA Gel Electrophoresis - Background Information

After isolation of RNA samples, the quality of the isolated RNA preparation is usually assessed by electrophoresis on a denaturing agarose gel. Although the results are mainly qualitative, you can get some information about the yield of the RNA as well.

Denaturing gels are used because RNA tends to fold upon itself and form extensive and stable secondary structure via intramolecular base pairing. This secondary structure of RNA prevents it from migrating mainly according to its size.

Make sure that you include an RNA positive control on the gel, in the case that you get unusual results you can then determine whether the problem was with the gel or was a problem with the RNA you are analyzing. You can also use RNA molecular weight markers, an RNA sample known to be intact, or both, can be used for this purpose.

RNA gels are visualized with ethidium bromide staining as ethidium bromide intercalates between the secondary structure of the RNA molecules and allows RNA to be seen under UV light.

RNA is separated out by gel electrophoresis (usually agarose gel electrophoresis). After running an RNA gel you can conduct a northern blot with subsequent transfer to membrane, hybridization with probe, and finally detection. Or simply (and much quicker), stain for RNA using ethidium bromide or SYBR (or similar dye - better as it is non-toxic and safer).

 

Applications of RNA Gels

As northern blots are much more tedious to complete than RNA gels and real-time PCR, they are not used as much as RNA gels.

The RNA gel electrophoresis and its variations are used in molecular biology research to:

  • detect RNA
  • assay for quality of RNA isolated
  • allow for general determination of RNA quantity or yield
  • assay for RNA degradation

Disadvantages of RNA Gels

The disadvantages of using RNA gel electrophoresis includes:

  • RNA gels are not very quantitative. You cannot determine precisely the RNA yield or amount even with standards. Radioactive methods such as northern blotting or dot blots are much more accurate.
  • Often ethidium bromide is used to detect the RNA. Ethidium bromide is a biohazard as it is mutagenic and toxic.
  • RNA gels allow for a quick determination of RNA yield however are not as fast as UV spectrophotometry or other methods.

Advantages of RNA Gels

The advantages of RNA Gels include:

  • It is a widely accepted method
  • RNA gels are very quick and easy to perform
  • You can assay for RNA quality within 1-2 hours
  • If you use SYBR or another non-toxic stain (i.e you do not use Ethidium Bromide), RNA Gels are quite safe.

RNA Gel Protocol

To verify the integrity of your RNA you should do a "Northern blot " analysis. In order to accomplish this you must run a denaturing gel.

For Mini-gels to Midi-gels of RNA use: Make 50 mL-100mL

For Mega-gels Make 400 mL.

 

For 100mL Gel (most commonly run) you need to add 1g of agarose to make 1% agarose solution.

 

Running Buffer Recipe for RNA Gels

To prepare: 500mls 750 mls 1.5 liters

1X E buffer (50X)-below
10 mL 15 mL 30 mL
0.66M formaldehyde (1/19 of vol) 26.3 mL 39.5 mL 79 mL

RNA Denaturing Reagents

To prepare 400ul of RNA denaturing reagent:

Add the following:

  • 80% Foramide-deionized 300ul
  • 3.7 % formaldehyde 40 ul
  • 1X 50X E buffer 8 ul
  • dH2O = 400ul 52ul

50 X E buffer per liter

Add:

  • 0.9 M Na2 HPO4 Dibasic 127.8 g
  • 0.1M NaH2PO4 Monobasic 13.8 g

Protocol: Steps in Preparing an RNA Gel

  1. put agarose in solutions.
  2. Heat prepared solution in tissue-plugged flask
  3. Let cool to 60°C
  4. Add Formaldehyde to equal 0.66M 2.7 ml 4.0 5.3 21.2
  5. (Add Ethidium Bromide if you are using it to flask)
  6. Pour gel (in hood if possible).
  7. Heat 2 ug of RNA at 75°C for 10 min., then quick cool. (this will allow RNA to denature and stay single-stranded).
  8. Add: 2.5 vol of RNA denaturing reagent
  9. Add 1/10 vol. of loading dye to samples.
  10. Load samples onto agarose gel wells.
  11. Run Gel (usually 100V volts) for 30minutes-2 hours. (Check RNA running using dye markers)


Tips for RNA Gels

Always run an agarose gel of your RNA to assess the quality. Do not only rely only UV spectrophotometry results.

* Use DEPC treated water and baked glassware for all the steps. The buffers must be Rnase free or use Milipore purified water if you are in a rush. Rnase is everywhere! Wear gloves, use only baked glassware, or virgin plastic. DePC treat your water, buffers (except Tris). Be very careful.

 

Troubleshooting RNA Gels

If you currently have a preferred method for running RNA, continue to use it.

Discuss and post problems or questions about RNA Gels in the RNA Protocols Forum.

RNA Gel References

 

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