|Bennett, Selester - NATURE DIAGNOSTICS|
Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 5, 2007
Publication Date: March 1, 2008
Citation: Miller, H.B., Ledbetter, C.K., Bennett, S. 2008. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels. Plant Biotechnology Journal. 3:360-363. Interpretive Summary: RNA extraction is, of itself, notoriously difficult. There are few RNA extraction protocols or commercial kits that work well with the starchy endosperm of cereal grains. There are, however, numerous commercial DNA extractions kits that are less expensive, often designed to minimize ending up with hazardous wastes to dispose, and 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 (white rice for consumption). We report the successful isolation of RNA from rice using 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, which codes for isopentenyl pyrophosphate isomerase (IpI), is 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 without leaving any hazardous wastes to be disposed.
Technical Abstract: Extraction of total RNA from starchy plant material such as common food grains is difficult, and especially so from dry rice (Oryza sativa L.) kernels. Most commercial RNA kits are not suited for starchy materials and traditional RNA extraction procedures leave hazardous organic wastes that have expensive disposal costs. On the other hand, commercial DNA extractions kits are less expensive, often designed to minimize disposal problems, and well-targeted for specific tissue types. Interestingly, the numerous commercial DNA kits now available often include directions for eliminating co-isolated RNA. This indicated a possibility of obtaining the generally unwanted RNA by-product by altering conditions to optimize for its isolation. We evaluated several commercial and standard methods that were promoted as being able to work under high polysaccharide conditions for RNA extraction from rice seeds. The target transcript for this work is the IpI (isopentenyl pyrophosphate isomerase) enzyme (EC 126.96.36.199). IpI is important in numerous isoprenoid pathways, including carotenoid synthesis. We are interested in the carotenoid synthesis enzymes present in the rice endosperm (milled rice) but needed to establish a constitutively expressed mRNA of that pathway before investigating the upregulation of other mRNAs. The presence of IPI (also variously written as Ipp, Ipi, and IppI) mRNA in the endosperm of mature white rice could indicate that carotenoid synthesis could occur in the normally carotenoid-free endosperm, provided transcription of the remaining machinery necessary for carotenoid biosynthesis were induced by environmental effects. We designed the primer for IPI to span an intron and thus produce a band at 511 bp with cDNA derived from mRNA but to produce a band at 730 bp when genomic DNA was amplified. Of the methods used, the Cartagen DNA Food Kit most successfully extracted DNA and RNA that produced cDNA from IPI using RT-PCR. When DNase was added, only the 511 bp mRNA cDNA product was produced. The sequence of the 511 bp cDNA product from the extraction by the Cartagen kit was directly sequenced and found to be 100% homologous to the IPI sequence for rice in the NCBI database. 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 without leaving any hazardous wastes to be disposed.