We have tested Invitrogen's SuperScript™ First-Strand Synthesis System and Ambion's RetroScript™ RT kit for cDNA synthesis from total RNA. Both kits produce high-quality cDNA templates for real-time PCR. Random primers are recommended in RT reactions because they provide full transcript
Real-time PCR primer validations for 16 cytochrome P450 genes.
A. PCR amplifications of 16 cytochrome P450 genes. Lane 1: 25 bp DNA ladder; lane 2-17: 10 |jl PCR products of P450 1a2, 2a5, 2b9, 2b13, 2c29, 2c38, 2c40, 2d26, 2d1, 2j5, 3a16, 3a25, 4a10, 4a12, 4a14, and 7a1.
B. Melting curves of 6 genes from cytochrome P450 family 1 and 2 (plotted as the first derivative of the absorbance with respect to temperature). Adapted from Nucleic Acids Research 31(24): e154 with permission.
coverage. The random priming approach gives highest detection sensitivity toward the 5' end of the sequence.
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