Worm Breeder's Gazette 15(3): 12 (June 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Dept. of Med. Tech., Kanagawa Pref. Coll. Nurs. & Med. Tech., Yokohama, Kanagawa 241, Japan, Dept. of Mol. Life Sci., Tokai Univ., Isehara, Kanagawa 259-11, Japan|
|2||Dept. of Med. Tech., Kanagawa Pref. Coll. Nurs. & Med. Tech., Yokohama, Kanagawa 241, Japan|
|3||Dept. of Mol. Life Sci., Tokai Univ., Isehara, Kanagawa 259-11, Japan|
In case of weakly expressed genes, Northern blot analysis may not be sensitive enough to detect transcription unless poly(A)+ RNA is prepared. We would like to report a simple method of RT-PCR assay from single animals that does not require poly(A)+ RNA preparation. Washed with M9 buffer, each single animal is transferred in 2 ul of TE (pH 8.0) in a 0.5 ml tube and frozen (-80oC, 20 min). 1 ul of lysis buffer [0.5 mg/ml proteinase K in 10 mM Tris (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 0.45% Tween 20 and 0.05% gelatin] is added and mixed well. After the addition of a mineral oil overlay, the tube is heated at 65oC for 1 hour and the reaction is stopped by the boil for 15 min. After cooling to 4oC, 1 ul of DNase I (7.5 units/ul, Pharmacia Biotech) is added and incubated at 37oC for 30 min. The reaction is stopped by boiling for 15 min. 46 ul of a master mix is then pipetted on top of the mineral oil overlay. The mix is formulated to bring the reaction volume to 50 ul using DEPC H2O with these final conditions: each 10 pmol of sense and anti-sense primers, 25 ul of 2x reaction mix and 1ul of SuperScript II RT/Taq DNA polymerase mix (GIBCO BRL). After a brief microfuge spin, the sample is incubated at 45-55oC for 30 min for cDNA synthesis. Then, 94oC for 2 min followed by amplification of 45 cycles of 94oC for 30 sec, 50-65oC (depending on the primer set) for 30 sec and 72oCo for 0.5-3 min (1 kb/min), followed by 72oC for 5 min. The PCR product (10 ul) is analyzed on 0.8-1.5% agarose gels. Using this method, we have been able to detect a typical sod-3 RT-PCR product of which the expression is lower than that of other genes. To avoid error in different stages, we recommend using some animals at the same developmental stage and duplicate with each primer set. This system is also useful for late-stage embryos. References: 1. Williams, B., D. et al. (1992) Genetics 131: 609-624 2. Andachi, Y. et al. (1993) WM'93: 33 3. Lee, E. H. et al. (1997) Focus 19 (2): 39-42 4. Sitaraman, K. et al. (1997) Focus 19 (2): 43-44