This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
CHAPTER 9 - MOLECULAR METHODS
A Simplified Ribonuclease Protection Assay for Embryos
(Source: G.M. Kelly and R.T. Moon)
This is a ribonuclease protection assay based on the methods of Thompson
and Gillespie (Anal. Biochem.163:281-291, 1987) and
Haines and Gillespie (Biotechniques 12:736-740, 1992). This rapid technique,
which eliminates the need to isolate purified RNA, is extremely sensitive such
that an overnight exposure is sufficient to detect a wnt1 signal from
ten 12h embryos (Kelly and Moon, in preparation). In addition, with the
generation of a standard curve, this assay can also be used to determine the
abundance of a specific transcript.
1. Collect embryos at particular developmental stages in 1.5 ml micro-
centrifuge tubes, and remove the medium. Add 45 µl of lysis buffer (4 M
guanidine thiocyanate, 25 mM sodium citrate and 0.5% sarcosyl) for every ten
2. Draw embryos and lysis buffer through a 22 gauge needle into a syringe,
expel and repeat three times, then vortex and store at -20°C.
3. Centrifuge the embryonic lysates for 1 min to pellet the broken chorions.
For hybridization, transfer 45 µl of each sample to tubes containing
1x105 cpm of 32P-UTP-labeled antisense probe
diluted in 5 Êl of lysis buffer. Varying the amount of labeled probe may be
necessary to optimize signal to background. We prefer the Maxiscript kit
available from Ambion (Austin, TX) for making the probes.
4. As a positive control and to generate a standard curve, hybridize 45
µl of lysis buffer containing either 1.0, 0.1, 0.01, or 0.001 ng of synthetic
sense strand RNA with the radioactive antisense probe. Similarly, hybridize the
antisense probe with 45 µl of lysis buffer containing 10 µl of
torula tRNA to ensure that the RNAse will digest any unprotected
single-stranded RNA molecules.
5. After an overnight hybridization at 55°C, mix the
sample with 500 µl of RNAse cocktail (20 µl RNAse A, Sigma R-5503,
previously boiled for 5 min, aliquoted and stored at -20°C plus 500 U of RNAse
T1, Sigma R-1003, in 10 mM Tris-HCl, pH 7.5, 300 mM NaCl and 5 mM
EDTA), and incubate at 37°C for 1 hr. Varying the hybrid-
ization temperature may have an effect of the sensitivity of the assay, we begin
at 55°C and then, if necessary, alter the temperature in 5°C intervals.
6. To isolate nucleic acids after hybridization, digest the lysate with 10
µl of 20% SDS and 5 µl of 20 mg/ml proteinase K, for 45 min at
37°C. Add Isopropanol (500 µl) and 3 ng of
torula RNA to each sample before centrifuging. Air dry the pellets,
resuspend in 10 µl of loading buffer (88% formamide, 10 mM EDTA, 1
mg/ml each of xylene, cyanol, and bromophenol blue), heat at 75°C for 4 min,
then place on ice before loading onto a 5% polyacrylamide/urea/taurine gel
(details in the Sequenase kit, US Biochemical). Electrophorese samples next to
2000 cpm of undigested probe and DNA sequencing reactions to provide size
markers, and process the gel for standard autoradiography.
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