RNA electrophoresis: Difference between revisions
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==Critical steps== | ==Critical steps== | ||
*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. | *RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient. <cite>LabGuidetoRNA</cite> | ||
*The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel. | *The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel. | ||
==Troubleshooting== | ==Troubleshooting== | ||
* | *RNases are the biggest problem in RNA work. [[Avoiding RNase contamination|Use proper precautions]]. | ||
==Notes== | ==Notes== | ||
*For best resolution, pour gels as thin as possible and run at low voltage. <cite>RNAmethodologies</cite> | *For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. <cite>RNAmethodologies</cite> | ||
*[[Tom Knight]] strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde. | *[[Tom Knight]] strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde. | ||
*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. <cite>MolecularCloning1</cite> | |||
== | ==RNA size markers / ladders== | ||
* [http://www.neb.com/nebecomm/products/productN0362.asp NEB ssRNA ladder $58], [http://www.neb.com/nebecomm/products/productN0363.asp dsRNA ladder $75, 25 lanes] | |||
* [http://www.fermentas.com/catalog/electrophoresis/rnaladders.htm RiboRuler RNA ladders from Fermentas $130/145 25 lanes] | |||
* [http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=15623200&messageType=catProductDetail Invitrogen 0.5-10kb RNA ladder €150 75µg] | |||
* [https://www.roche-applied-science.com/servlet/RCProductDisplay?productId=3.6.12.4.5.1 Roche DIG-labelled RNA ladder €160 2µg] | |||
* [http://www.protocol-online.org/biology-forums/posts/12166.html short discussion on using DNA as size marker for RNA gels] | |||
==Specific Protocols== | |||
*[[Jacobs:Protocol RNA Agarose Gel]] | |||
*[[Knight:RNA electrophoresis/Native]] | |||
*[[Jacobs:Protocol Agarose Formaldehyde Gel Preparation for RNA Electrophoresis]] | |||
*[[Knight:RNA electrophoresis/Denaturing]] | |||
*[[Endy:Northern Blot, 32P End-Labeled Probes#Process the Gel (3-4 hours)]] | |||
==References== | ==References== | ||
<biblio> | <biblio> | ||
# | #MolecularCloning1 [[doi:10.1101/pdb.prot4057|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]] | ||
#MolecularCloning2 [[doi:10.1101/pdb.prot4050|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde]] | |||
#RNAmethodologies isbn=0-12-249695-7 | #RNAmethodologies isbn=0-12-249695-7 | ||
#CurrentProtocols Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment | #CurrentProtocols [[doi:10.1002/0471142727.mb0409s67|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]] | ||
#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion | |||
#LabGuidetoRNA isbn=0-471-12536-9 | |||
#ProtocolsOnline [http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html RNA electrophoresis] | |||
</biblio> | </biblio> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:RNA]] | [[Category:RNA]] | ||
[[Category:In vitro]] | [[Category:In vitro]] | ||
Latest revision as of 07:33, 2 March 2009
Curators
Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute.
Abstract
Electrophoresis permits assessment of RNA by size and amount. In general, electrophoresis of RNA is done as a step prior to Northern analysis.
Materials
List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc. Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
Reagents
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
Equipment
Any equipment used to perform the protocol (link to a method for using them).
Procedure
A step by step guide to the experimental procedure.
Critical steps
- RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient. [1]
- The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.
Troubleshooting
- RNases are the biggest problem in RNA work. Use proper precautions.
Notes
- For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. [2]
- Tom Knight strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde.
- Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. [3]
RNA size markers / ladders
- NEB ssRNA ladder $58, dsRNA ladder $75, 25 lanes
- RiboRuler RNA ladders from Fermentas $130/145 25 lanes
- Invitrogen 0.5-10kb RNA ladder €150 75µg
- Roche DIG-labelled RNA ladder €160 2µg
- short discussion on using DNA as size marker for RNA gels
Specific Protocols
- Jacobs:Protocol RNA Agarose Gel
- Knight:RNA electrophoresis/Native
- Jacobs:Protocol Agarose Formaldehyde Gel Preparation for RNA Electrophoresis
- Knight:RNA electrophoresis/Denaturing
- Endy:Northern Blot, 32P End-Labeled Probes#Process the Gel (3-4 hours)
