小中大我现在用B-actin做为RT-PCR的内参,是不是可以用楼上的引物序列啊?(
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不过SCIENCE那篇文章的引物在PRIMER中的PRODUCT RATING比PRIMER搜到的引物得分高(51)。我还有一对ROCHE公司的引物，如下：
Comparison of C. therm. Polymerase One-Step RT-PCR System and Tth DNA Polymerase
The C. therm. Polymerase One-Step RT-PCR System was compared with Tth DNA Polymerase for ability to amplify limiting amounts of a 324 bp b-actin sequence from mouse RNA. Total RNA was isolated from mouse liver and used for one-step RT-PCR.
Each reaction mix (50 µl total volume) contained template RNA (0,1 ng to 100 ng mouse liver RNA), enzyme (either 6 units C. therm. Polymerase mix, 5 units Tth DNA Polymerase, or 15 units Tth DNA Polymerase), reverse and forward PCR primers, PCR Nucleotide MixPLUS and buffer components recommended for optimal enzyme activity. The forward (5'-AGC TTG CTG TAT TCC CCT CCA TCG TG-3') and the reverse primer (5'-AAT TCG GAT GGC TAC GTA CAT GGC TG-3') were designed to amplify a 324 bp sequence from b-actin mRNA. The RT reaction was performed at 60°C for 30 min, followed by PCR in a Perkin Elmer Model 9600 Thermal Cycler with the following thermal profile:
· Initial denaturation: 94°C, 2 min
· 10 cycles of denaturation (94°C, 30 s),
annealing (60°C, 30 s), elongation (68°C, 60 s)
· 25 cycles of denaturation (94°C, 30 s), annealing (60°C, 30 s), elongation (68°C, 60 s + 5 s/cycle)
· Final extension: 68°C, 7 min.
它的得分是42，低于SCIENCE的引物，高于PRIMER搜到的引物，也可考虑使用。
