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RNA Ladders: Introduction, Range Selection Guide, Reference Guide

• Introduction: RNA Electrophoresis
  • Fermentas Products for RNA Electrophoresis
  • Native RNA Electrophoresis
  • Denaturing RNA Electrophoresis
  • Assessment of RNA Quality
  • RNA Quantification
• Introduction: RNA Ladders
• Range Selection Guide
• Reference Guide

Introduction: RNA Electrophoresis

RNA molecules can be analyzed both on native or denaturing agarose and polyacrylamide gels. Native RNA electrophoresis eliminates the need for hazardous chemicals, but, due to intramolecular interactions, RNA molecules can be prone to forming extensive double-stranded structures, which may be quite difficult to disrupt. Native RNA electrophoresis is typically used to asses the overall quality of total RNA.
To determine the quality of RNA preparations more precisely, based predominantly on the size of the molecules, RNA is separated in the presence of strong denaturants, hence denaturing RNA electrophoresis.

Fermentas offers following products for RNA electrophoresis:

• New RiboRuler™ Low Range RNA Ladder and RiboRuler™ RNA Ladder, High Range. These ladders are mixtures of chromotography purified RNA transcripts and are ideal for ssRNA sizing both on native and denaturing gels. RNA concentration in Fermentas RNA ladders is measured spectrophotometrically. Purification of each RNA transcript by chromatography ensures that ladders are free of NTP’s, and are suitable for RNA quantification on gels.
  RNA ladders are available in ready-to-use and conventional formats. Conventional versions can be labeled radioactively and are ideal for Northern blots.
• 2X RNA Loading Dye is used for RNA probes preparation prior to electrophoresis. The solution contains an optimal concentration of formamide what allows for separation of RNA molecules according to their size even under non-denaturing conditions. The presence of ethidium bromide in the solution allows for RNA visualization without additional staining of denaturing agarose gels.
• 50X TAE and 10X TBE buffers are free of ribonucleases and can be used for RNA gel electrophoresis. 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.
• TopVision™ LE GQ Agarose and TopVision™ LM GQ Agarose are free of ribonucleases and can be successfully used for analytical and preparative RNA electrophoresis.

Native RNA Electrophoresis
Native RNA electrophoresis can be performed in Fermentas 50X TAE Buffer or 10X TBE Buffer and TopVision™ LE GQ Agarose gel prepared in the same buffer. For optimal results ethidium bromide should be present in both the native agarose gel and the electrophoresis buffer at a final concentration of 0.5 µg/ml.

Denaturing RNA Electrophoresis
Frequently used conditions for denaturing RNA electrophoresis are the following:

• Denaturing polyacrylamide gel electrophoresis in TBE buffer supplemented with 7 M urea. Fermentas RiboRuler™ Low Range RNA Ladder and 5% polyacrylamide-urea gel is recommended for analysis of smaller RNA molecules.
• Formaldehyde agarose gel electrophoresis in MOPS buffer. Formaldehyde forms unstable Schiff bases with the imino-groups of guanine residues. These adducts maintain RNA in the denatured state by preventing intra-molecular Watson-Crick base paring.
• Glyoxal/DMSO agarose gel electrophoresis in sodium phosphate buffer. The two aldehyde groups of glyoxal react under slightly acid conditions with the imino-groups of guanine to form cyclic derivatives that prevent the intra-strand Watson-Crick base paring.

Assessment of RNA Quality
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 can 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. No band smearing of either band should occur.
The band of 28S human rRNA migrates approximately like the 5000 b band, the band of 18S human rRNA migrates approximately like the band of 1900 b.

RNA Quantification

New RiboRuler™ RNA ladders are suitable for RNA quantification on gel. Exceptionally pure ladders are produced from chromatography-purified RNA transcripts and are free from any degraded RNA or NTP’s that very often interfere with spectrophotometric measurements and lead to an erroneous RNA quantification. Spectrophotometrically determined RNA concentrations of ladder bands are given here.

Introduction: RNA Ladders

For an accurate single-stranded RNA sizing and for RNA quantification on gels Fermentas offers:
RiboRuler™ Low Range RNA Ladder (100-1000 b)
RiboRuler™ High Range RNA Ladder (200-6000 b)

They form sharp bands of uniform intensity and have easy-to-remember RNA fragment sizes.
The ladders can be stained with ethidium bromide or by other staining techniques.
The RNA concentration of each ladders band is determined spectrophotometrically and given in the Certificate of Analysis of the product.
Conventional versions of Fermentas RiboRuler™ RNA ladders are suitable for end-labeling with T4 Polynucleotide Kinase and are ideal for Nothern blots.
All Fermentas RNA ladders are supplied with 2X RNA Loading Dye.
Ready-to-use RNA ladders are premixed with RNA Loading Dye and can be applied directly onto native or denaturing agarose and polyacrylamide gels.
RiboRuler™ RNA ladders are provided in small aliquots (5x10 µg) to minimize the possibility of contamination and degradation during the usage and multiple freeze-thaw cycles.
Besides standard assay procedures, the exceptional purity of Fermentas RNA ladders is confirmed by analysis on an Agilent 2100 bioanalyzer (see picture below).

Analysis of RiboRuler™ RNA Ladder, High Range, using an Agilent 2100 bioanalyzer, RNA 6000 Nano LabChip® Kit and agarose gel electrophoresis. A - analysis of RiboRuler™ High Range RNA Ladder on Agilent 2100 bioanalyzer B - agarose gel analysis of RiboRuler™ High Range RNA Ladder

Range Selection Guide

Reference Guide

Updated vasario 01, 2008 09:52