Knight:RNA electrophoresis/Native

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<font color=red>in progress!</font>
 
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==Overview==
==Overview==
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Electrophoresis permits assessment of RNA by size and amount.  However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure.
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Electrophoresis permits assessment of RNA by size and amount.  However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure.  Since this is a native gel you are more likely to see a smear of RNA and/or multiple bands because of RNA secondary structure.
==Procedure==
==Procedure==
The procedure for running a native RNA gel is very similar to that for running a [[Knight:Agarose gel electrophoresis|DNA agarose gel]].  Here are a few things to note.
The procedure for running a native RNA gel is very similar to that for running a [[Knight:Agarose gel electrophoresis|DNA agarose gel]].  Here are a few things to note.
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#At least some protocols discuss using TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA).  However, TAE buffer should be sufficient?
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#Generally load 1 &mu;g and 2.5 &mu;g samples.
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#The samples can be loaded directly without heating.
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#Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?
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#*''TAE buffer also seems to work equally well in my hands for ~1000bp transcripts.''
#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
#An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
#Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
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#*''I tried ~65V total.''
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#Use an RNase free aliquot of [[Knight:Loading dye|loading buffer]] (i.e. made with RNase free water).
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#RNA Ladder
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#*2 &mu;L [http://www.neb.com/nebecomm/products/productN0362.asp RNA ladder]
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#*2 &mu;L loading dye
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#*8 &mu;L H<sub>2</sub>O
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#Also run a DNA ladder
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===Staining===
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#Dilute SYBR Gold 10,000-fold into 1X running buffer. 
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#*Note that SYBR gold is preferable to SYBR green II
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#Incubate agarose gel in staining solution for 40 minutes.
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#Visualize on gel imager. (No destaining needed).
==Notes==
==Notes==
*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis.  The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>
*''These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis.  The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility.'' <cite>ProtocolsOnline</cite>
*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>
*''Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures.'' <cite>Ambion</cite>
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*''TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 nt RNA and for denaturing polyacrylamide gel electrophoresis.'' <cite>Fermentas</cite>
==References==
==References==
#ProtocolsOnline [http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=842 Gel Electrophoresis of RNA - general]
#ProtocolsOnline [http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=842 Gel Electrophoresis of RNA - general]
#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA]
#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA]
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#Fermentas [http://www.fermentas.com/catalog/electrophoresis/rnaguide.htm Introduction: RNA Electrophoresis]
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:In vitro]]
[[Category:In vitro]]
[[Category:RNA]]
[[Category:RNA]]

Current revision

Contents

Overview

Electrophoresis permits assessment of RNA by size and amount. However, in native gels RNA electrophoretic mobility will depend on not only the size of the RNA but also its secondary structure. Since this is a native gel you are more likely to see a smear of RNA and/or multiple bands because of RNA secondary structure.

Procedure

The procedure for running a native RNA gel is very similar to that for running a DNA agarose gel. Here are a few things to note.

  1. Generally load 1 μg and 2.5 μg samples.
  2. Use TBE buffer (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA)?
    • TAE buffer also seems to work equally well in my hands for ~1000bp transcripts.
  3. An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
  4. Run the gel at low voltage (reports of 5-6 V/cm or 8V/cm are common as measured between the electrodes).
    • I tried ~65V total.
  5. Use an RNase free aliquot of loading buffer (i.e. made with RNase free water).
  6. RNA Ladder
  7. Also run a DNA ladder

Staining

  1. Dilute SYBR Gold 10,000-fold into 1X running buffer.
    • Note that SYBR gold is preferable to SYBR green II
  2. Incubate agarose gel in staining solution for 40 minutes.
  3. Visualize on gel imager. (No destaining needed).

Notes

  • These are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility. [1]
  • Native agarose gel electrophoresis may be sufficient to judge the integrity and overall quality of a total RNA preparation by inspection of the 28S and 18S rRNA bands. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Bands are generally not as sharp as in denaturating gels, and a single RNA species may migrate as multiple bands representing different structures. [2]
  • TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used for smaller than 1500 nt RNA and for denaturing polyacrylamide gel electrophoresis. [3]

References

  1. ProtocolsOnline Gel Electrophoresis of RNA - general
  2. Ambion Agarose Gel Electrophoresis of RNA
  3. Fermentas Introduction: RNA Electrophoresis
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