DNase concentration assay to obtain DNA-free RNA from sugarcane leaves

• Published in: Genetics and molecular research Vol. 15; no. 4
• Main Authors: Santos, J A, Luz, G A, Oliveira, K P, Oliveira, L F, Andrade Júnior, A S, Valente, S E S, Lima, P S C
• Format: Journal Article
• Language: English
• Published: Brazil 02.12.2016
• Subjects: Deoxyribonucleases - metabolism; DNA - metabolism; Gene Expression; Plant Leaves - genetics; RNA, Plant - analysis; RNA, Plant - standards; Saccharum - genetics
• ISSN: 1676-5680; 1676-5680
• DOI: 10.4238/GMR15049073

The success of gene expression studies, protein synthesis, and construction of cDNA libraries directly depends on the purity and integrity of the RNA used in these tests, as even minor amounts of contaminant DNA (<1%) can produce a false positive amplification signal in quantitative real-time PCR. For RNA contaminated with genomic DNA, an essential step in the studies on gene expression is the treatment of the RNA samples with DNase. This study was conducted to test three different concentrations of DNase I (0.02, 0.04, and 0.06 μL/​​ng of RNA), which were chosen based on the results of the RNA sample quantifications and as indicated by the manufacturer, to digest genomic DNA present in the RNA samples extracted from sugarcane leaves with the Concert™ Plant RNA Reagent. The results showed that all three concentrations of DNase significantly reduced DNA concentrations. However, RNA was also degraded on DNase I treatment. In addition, the amount of DNA present in the RNA samples after purification with DNase I was sufficient for its amplification in the tests conducted with conventional PCR. Furthermore, the condition of RNA samples obtained after the treatments allowed for real-time PCR. Therefore, we concluded that 0.02 μL DNase I was the ideal concentration for sugarcane RNA purification, as higher concentrations do not increase the efficiency of the genomic DNA digestion in RNA samples and only make the purification process more expensive. This study provides important information on the effect of high concentrations of DNase I and complements previous studies that have so far tested only the DNase concentration recommended by the manufacturer.