Invited Symposium: Molecular Mechanisms of Ageing
Materials and Methods
Materials and Methods Synaptoneurosome Preparation Synaptoneurosomes were isolated from the cortex of 12 day-old Long-Evans rats by sequential syringe filtration through nylon membranes of decreasing pore size followed by low speed centrifugation 1. RNA was prepared using TRI-ReagentTM according to the manufacturer1s directions. Contaminating genomic DNA was removed by precipitation LiCl. Differential Display DDRT-PCR was carried out according the procedure of Sung and Denman to minimize false positive clones 2. One microgram of RNA was converted to first strand cDNA by mixing 10 ml RNA with 2.5 ml of 20 mM of one of three anchored 31 PCR primers (Table 1.) and 9.5 ml of DEPC-H2O. Subsequently, a master mixture consisting of 5 ml 5X reverse transcriptase buffer, 1 ml (40 Units) of RNAsin, 2.5 ml of 0.1 mM DTT, 2.5 ml of 0.25 mM dNTPs and 1 ml (200 Units) of reverse transcriptase was added; the sample was then incubated for 70 min. at 37oC. To produce the differential display, one microliter of each reverse transcription mixture was amplified in the presence of [a-32P] dCTP. Each amplification sample consisted of the following components: 2 ml of 10X Taq DNA polymerase buffer, 1.5 ml of 0.1 mM dNTPs, 1.25 ml (20 mM) of one of six 51 primers (Table 1.), 1 ml of a one-to-five dilution of [a-32P] dCTP (3000 Ci/mmol), 0.5 ml of Taq DNA polymerase and 12.75 ml of DEPC-H2O. The reaction mixtures were amplified using a 25 cycle paradigm consisting of a 94oC, 1.5 min. denaturing step, 42oC, 30 sec. and 57oC, 1 min. annealing steps, and a 72oC, 1.5 min. extension step. Following the reaction, 5 ml of 90% formamide, 20 mM EDTA, 0.3% xylene cyanole and 0.3% bromphenol blue was added; each sample was then boiled for 3 min. Five microliters of the mixture was loaded on to 6% polyacrylamide 7M urea sequencing gels and electrophoresed for 2-3 hr at 1500 V. Subsequently, the gels were transferred to Whatman 3M chromatography paper and dried; differentially displayed bands were detected on Kodak XAR5 film after 1-5 hr of exposure.
The resulting cDNA was amplified with 5 pmol of each primer, 1.25 U of Taq DNA polymerase and 5 nmol of each dNTP in 25 ml using the following cycling paradigm:
Fig. 1: Touchdown RT-PCR.
Various concentrations of cDNA were assessed to insure the amplification of each amplicon was in the linear range.
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|Sung, Y-J.; Weiler, I. J.; Greenough, W. T.; Denman, R. B.; (1998). Selectively Enriched Messenger RNAs in Rat Synaptoneurosomes. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/higuchi/sung0691/index.html|
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