Molecular Techniques and Methods

Analysis of RNA Quality

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

RNA quality can be checked by electrophoresis in agarose gel, by electrophoresis in polyacrylamide gel, or by in vitro translation.




ELECTROPHORESIS OF RNA IN AGAROSE GEL

RNA samples (approximately 1 ug) can be subjected to electrophoresis through l.0% agarose gel and stained with ethidium bromide (1 ug Et-Br/ ml). This method is very fast and allows a gross indication of the presence of both 25 and 16 S ribosomal RNA peaks.


Preparation of Agarose Gel (100 ml)
Agarose ------------------------------------- 1 g
40 mM Tris-HCl (pH 8.0) ------------------- 4 ml of 1 M Tris-HCl
20 mM Hydrogen acetate -------------------- 2 ml of 1 M Hydrogen acetate
2 mM EDTA (pH 8.0) ----------------------- 0.4 ml of 0.5 M EDTA
DEPC-treated H2O -------------------------- 92.6 ml





ELECTROPHORESIS OF RNA IN POLYACRYLAMIDE GEL

RNA samples (2-10 ug) can be subjected to electrophoresis through either cylindrical or slab 2.5 % polyacrylamide gels, and subsequently scanned at 260 nm with an ISCO gel scanner. Although laborious, this is the most reliable method for checking RNA integrity, since it will show very limited degradation resulting in a shift in relative abundance between the 25 and 16 S ribosomal RNA peaks.


Preparation of 2.5 % Polyacrylamide Gel (100 ml)
30% Acrylamide:Bis Solution (29:1) ------- 8.4 ml
40 mM Tris-HCl (pH 8.0) ------------------- 4 ml of 1 M Tris-HCl
40 mM NaH2PO4 --------------------------- 4 ml of 1 M NaH2PO4
2 mM EDTA (pH 8.0) ----------------------- 0.4 ml of 0.5 M EDTA
0.2% SDS ----------------------------------- 2 ml of 10% SDS
10% Glycerol -------------------------------- 20 ml of 50% Glycerol
DEPC-treated H2O -------------------------- 61.2 ml

  • Add 750 ul of 10% ammonium persulfate and 85 ul of TEMED.
    Pour the gel mixture slowly into the previously prepared gel form, until the liquid level reaches the top of the upper glass plate.


  • Insert the comb between the two glass plates and clamp in place with a small spring clip.


  • Polymerize the gel for 1 hour.





  • In vitro TRANSLATION

    By comparing incorporation levels to known RNA standards of good quality, the integrity of isolated mRNA can be checked. The in vitro system is very sensitive to mRNA degradation, resulting in an immediate drop in incorporation level. However, although this method is very fast (3-4 hours), it is also expensive, necessitating radioactively labeled amino acids. In addition, if impurities of the RNA (polysaccharides, or salt) are present, this will result in reduced incorporation.



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