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.
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.