ISOLATION OF HIGH-QUALITY RNA FROM APPLE (MALUS X DOMESTICA) FRUIT

Authors: ASIF, MEHAR - UNIVERSITY OF MARYLAND; TRIVEDI, PRABODH - NATL BOTAN RES INST INDIA; SOLOMOS, THEOPHANES - UNIVERSITY OF MARYLAND; Tucker, Mark

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/23/2006
Publication Date: 5/23/2006
Citation: Asif, M., Trivedi, P., Solomos, T., Tucker, M.L. 2006. Isolation of high-quality rna from apple (malus x domestica) fruit. Journal of Agriculture and Food Chemistry. 54:5227-5229.

Interpretive Summary: Isolation of RNA is essential to perform molecular experiments. Nonetheless, after all these years there are still some plant tissues that remain difficult to extract RNA from using standard procedures. We have developed and tested two procedures that utilize an initial ethanol or acetone wash of frozen crushed plant tissue that eliminates the interfering compounds. We have tested these procedures using a plant tissue that has historically been difficult to extract RNA from, i.e., ripe apple fruit. We demonstrated that both of these procedures yield high quality RNA and that the acetone washed homogenate routinely produced a significantly higher RNA yield than the ethanol wash procedure. For those scientists working on plant tissues and organs that have proven difficult for RNA isolation these procedures have the potential to improve the yield and quality of the extracted RNA.

Technical Abstract: It is difficult to isolate sufficient quantities of high quality RNA from apple fruit. An abundance of polyphenolic compounds and polysaccharides and a relatively low concentration of RNA in the fruit tissue create conditions that make RNA isolation difficult with standard techniques. We have developed two RNA isolation methods that include an initial homogenization and extraction with acetone or ethanol that remove the interfering compounds and precipitate the protein and nucleic acids for subsequent RNA extraction. RNA was extracted from the prepared powders with 100 mM Tris-HCl (pH 8.0) containing 2% CTAB, 20 mM EDTA, 1% '-mercaptoethanol and 1.4 M NaCl. The quality of the extracted RNA was then assessed spectrophotometrically by determining the absorbance ratio at 260 nm and 280 nm, RT-PCR, and Northern blot analysis. The quality of RNA was satisfactory with both acetone and ethanol preparations; however, the acetone powder produced consistently higher quantities of RNA.