Using a Commercial DNA Extraction Kit to Obtain RNA for RT-PCR from Starchy Rice Endosperm.
Helen Belefant-Miller, USDA-ARS, Dale Bumpers National Rice Research Center, 2890 Hwy 130 E., Stuttgart, AR 72160
Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that are less expensive than, produce few hazardous wastes, and are well-targeted for specific tissue types. Most of these kits note that the DNA product will contain significant amounts of RNA unless an RNase step is included. Our intent was to take advantage of the presence of the usually unwanted RNA by-product to develop a simple, rapid, inexpensive, and effective means of isolating intact RNA from a particularly recalcitrant tissue, starchy rice (Oryza sativa L.) endosperm. We report the successful isolation of RNA from rice by a modification of the Cartagen DNA Food Extraction kit. Success was determined by the production and amplification of cDNA by reverse transcription PCR (RT-PCR) of IPI. IPI mRNA codes for isopentenyl pyrophosphate isomerase (IpI), an early enzyme in the carotenoid biosynthesis pathway. By optimizing the Cartagen kit for the isolation of the RNA by-product, we have found a way to quickly isolate RNA from rice endosperm inexpensively without leaving any hazardous wastes to be disposed.