XB-IMG-158089
Xenbase Image ID: 158089
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Fig. 3. An/a (a) and An/b (b) mRNAs are present throughout early
embryogenesis and can be detected at much lower levels in adult tissues,
RNase protection assays using transcript-specific probes are shown.
Protected products were separated on a 5% polyacrylamide/7 M urea
gel. The undigested An/a probe (P) is 395 nt; RNase digestion generates
a 279-nt protected fragment. The undigested Anlb probe (P) is 426 nt
long; RNase digestion generates a 376-nt protected fragment. Both
probes were derived from the 3’ untranslated regions. Panel A shows
RNase protection analysis of three egg/embryo equivalents (about 5
ug) of total RNA isolated from various stages of Xenopus development
[egg (E) through stage 241. Panel B shows RNase protection analysis
of total RNA isolated from various adult tissues: brain (B), gut (G),
heart (H), kidney (K), liver (L), muscle (M), lung (P), skin (S), testes (T),
stage V/VI oocyte (0). Total RNA (15 ug) was used in each assay,
except for 7.5 ug of oocyte RNA. The 18s and 28s ribosomal RNA (r)
bands are shown below the protected bands as a control for RNA
integrity. Methods: The antisense Anla probe used was an in vitro
synthesized T7 transcript made from the plasmid pT3R-21 digested
with AJIII. The antisense Anlb probe was a T7 transcript made from
a Hind111 digest of pE3LAB, a plasmid constructed by removing a 420-
bp fragment from the 3’ end of the insert in pE3L-4 by a BamHI + &/II
double digestion followed by religation. Transcription reactions were
performed as described earlier (Krieg and Melton, 1987). All RNA
probes were purified on a 5% polyacrylamide/7M urea gel prior to use.
All RNA was extracted as previously described (Chomczynski and
Sacchi, 1987). RNase protections were performed as described before
(Krieg and Melton, 1987; Dagle et al., 1990) with minor modifications
as outlined below. Total RNA extracted from eggs, staged embryos
(Nieuwkoop and Faber, 1967), or adult tissues was precipitated with
l-5 x 10’ cpm of gel purified “P-labeled RNA probe. The RNA-probe
pellet was washed with 75% ethanol and resuspended in 30 ul of 80%
deionized formamide/ mM l,4-piperazine-diethanesulfonic acid
(Pipes) pH 6.4/400 mM NaCl/l mM EDTA. The solution was heated
to 85°C for 5 min, then incubated for at least I2 h at 45°C. Following
this incubation, 350 pl of RNase digestion buffer (IO mM TrisHCl pH
7.4/300 mM NaCI/S mM EDTA) containing 40 pg/ml RNase A and
100 U/ml RNase T, was added on ice. After 90 min at 4°C IO ul of
20% SDS and 2.5 ul of 20 mg/ml proteinase K were added. Following
a IO min incubation at 37°C. the solution was extracted with an equal
volume of phenol:chloroform (5O:SO)T. he aqueous phase was precipitated
and resuspended in 50% formamide/lO mM EDTA/0.025% bromophenol
blue/0.025% xylene cyanol. The samples were heated for 5
min at 85°C before electrophoresis on a 5% polyacrylamide/7 M urea
gel. The gel was dried and exposed at -70°C to Kodak XAR-5 film. Image published in: Linnen JM et al. (1993) Copyright © 1993. Image reproduced with permission of the Publisher.
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