Abstract
Blueberry leaves are rich in antioxidants, and are expected to be useful as a material of functional foods. We have made DNA markers related to polyphenol synthesis genes by cDNA subtraction, and have tried to select cultivars including many polyphenols using DNA markers. It is difficult to isolate RNA from blueberry leaves because the leaves include many polyphenols. We established an efficient method for isolating RNA from blueberry leaves by a modification of the cetyl trimethyl ammonium bromide (CTAB) method. We isolated RNA from blueberry leaves using eight different buffers: borate, HEPES, MES, MOPS, PIPES, TES, Tricine and Tris. We obtained about 80 μg of RNA from 1g of blueberry leaves with a widely used Tris buffer. We obtained more than 200μg of RNA from 1 g of blueberry leaves with a HEPES or a MOPS buffer. The amount of RNA was sufficient for cDNA synthesis. We succeeded at performing cDNA synthesis and increasing the cDNA using polymerase chain reactions. We constructed a number of cDNA subtraction libraries, and have recently isolated cDNA fragments related to polyphenol synthesis genes from these libraries.
