Improved methods for storing and extracting DNA from Ilex paraguariensis (Aquifoliaceae) tissue samples
Mate (Ilex paraguariensis) leaves are popular for consumption as an infusion, which provides various health benefits due to its nutraceutical properties. Leaf samples oxidize after harvesting, requiring special handling to avoid DNA damage or degradation by enzymatic or oxidative activities. The objectives of this work were to evaluate several methods for sample storage and DNA extraction to identify practical and efficient protocols to guarantee the DNA quantity and integrity for molecular studies of mate. Total DNA was extracted from fresh leaves and compared with DNA extracted from leaves stored in silica gel at room temperature for 14 days or in CTAB (cetyltrimethylammonium bromide) buffer. The leaves were cleaned with absorbent paper and stored in 50 mL Falcon tubes containing approximately 25 g of silica gel or in 2 mL Eppendorf tubes containing approximately 1 mL of CTAB buffer. Samples treated with silica gel were stored at room temperature for 14 days, and the ones with CTAB buffer were stored either at 4°C for 14 and 90 days or at room temperature for 90 days. The DNA was quantified using a Nanodrop spectrophotometer and agarose gel electrophoresis. DNA purity (with regard to the presence of enzyme inhibitors) was tested by PCR amplification of fragments of the plastid gene, trnL-trnF. Samples of mate leaves can be stored on silica gel or in CTAB buffer for up to 90 days at room temperature without reduction in DNA quality. Samples stored in CTAB buffer can be refrigerated at 4ºC to minimize oxidation of phenolic compounds. The improved methods for sample storage and DNA extraction with CTAB maintain quantity and integrity for conducting molecular studies of mate.