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YOSHITAKA HISHIKAWA ET AL.
Evaluation of results
Positive control
Negative control
RNase treatment
Competitive examinsation
28S rRNA complementary probe
Sense probe
RNase-A
Excess amount of either homologous or heterologous unlabeled
oligo-DNA
Sense probe should be used as a negative control, and we use 28S rRNA
probe as a positive control to evaluate the levels ofhybridizable RNAs in
tissue sections in every run.
Materials
for T-T - Oligo-deoxynucleotide (OligO-DNA)
dimerization
Oligo-DNA probe: In the case of the T-T dimer method, two
or three TT A repeats are added to both the 5' and 3' ends of the
original sequence and later dimerized by UV irradiation.
Design of Oligo-DNA probe
5'-TTATTA-Oligo-DNA (45 mer)-ATTATTATT-3'
** **
** **
**
* = T -T dimerizable regions by UV irradiation
- Melting temperature (Tm): Tm = 81.5 + 16.61l0g[Na+] + OAl(O/OGC)675/L [Na+] = the concentration of sodium cation; %GC = (guanine +
cytosine)/all bases; L = the probe length (bases)
- UV lamp (254 nm wave length) (Toshiba GL-15; Toshiba Ltd.,
Tokyo, Japan) UV radiometer (taking an accurate measurement of 5 J/m2/sec) : UVR-254 (Tokyo, kougaku Kikai, Ltd.,
Tokyo, Japan)
Note: For the UV source, we used two UV 15 W Toshiba GL-15
(Toshiba Ltd., Tokyo, Japan) lamps placed 36.1 em from the surface of the quartz dish. Instead of UV lamp and radiometer, uvcrossrinker (UV -stratalinker, stratagene cloning systems) is
available.
- flat siliconized quartz dish: Type AB, Gaskuro Kogyo Inc. Tokyo, Japan