Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm.
The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes.
Belefant-Miller
Helen
H
USDA-ARS Dale Bumpers National Rice Research Center, Stuttgart, AR 72160, USA. Helen.Miller@ars.usda.gov
Ledbetter
Cindy
C
Bennett
Selester
S
D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D012275:Oryza / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D052616:Solid Phase Extraction / Q000379:methods
D018749:RNA, Plant; D011933:Reagent Kits, Diagnostic; D004247:DNA