Comparison of RNA extraction methods in order to optimize total RNA extraction from borage tissues under cadmium stress

Abstract

IntroductionTranscriptomics studies speed up the basic and applied research on the identification of genes involved in the biosynthesis of medicinally significant primary and secondary metabolites as well as plant responses to biotic and abiotic stresses. The adequate quality and quantity of RNA are essential for successful transcriptomics investigations such as RNA sequencing (RNA-seq) and microarrays. It is extremely difficult to isolate RNA from medicinal plants with high levels of polyphenols and polysaccharides, such as Borago officinalis. Moreover, isolating nucleic acids from tissues exposed to stressful conditions of heavy metal toxicity such as cadmium is challenging due to the increased accumulation of reactive oxygen species (ROS) and secondary metabolites. Any RNA-seq experiment requires high-quality RNA because the isolated RNA should meet stringent quality control requirements in order to be sequenced on the various platforms. In the present study, we evaluated different RNA extraction methods to obtain an efficient protocol for isolating high-quality total RNA from borage tissue exposed to cadmium stress.Materials and methodsThe borage seedlings were grown in hydroponic containers containing half-strength Hoagland's nutrient solution in a growth chamber. Borage seedlings were exposed to 162 μM Cd using cadmium nitrate (Cd (NO3)2.4H2O) at 5-6 leaves stage and sampled at 48 h after treatment. The roots and leaves were subjected to five RNA isolation methods, including phenol/chloroform-based method, CTAB-based method, SDS-based method, RNX-plus protocol, and modified RNX-plus method to obtain an efficient protocol for isolating high-quality total RNA. The concentration and purity of the RNAs extracted using the abovementioned protocols were determined using gel electrophoresis and NanoDrop spectrophotometer. The quality and integrity of selected total RNA were approved with cDNA synthesis, RT-PCR, Bioanalyzer System, and transcriptome sequencing. After evaluating the extraction methods, a quick, simple and efficient instruction based on the modified RNX-Plus extraction method was afforded.Results and discussionThe results showed that the modified RNX-plus method was a fast and efficient protocol for the isolation of RNA from the borage leaf and root when compared with other methods. The method overcame the limitations posed by poor quality and low concentration of isolated RNA from borage samples exposed to cadmium stress. The A260/A280 and A260/A230 ratios of the RNA extracted using the modified RNX-plus method were 2.1 and 2.07, respectively, revealing its high purity. The key factors in the optimized protocol that resulted in removing the impurities were included the increasing ratio of extraction buffer to the amount of the powdered plant sample, using the optimized volume of chloroform, raising the RNA precipitation time at -20°C, washing RNA with lithium chloride and washing again with ethanol. Also, the yields of 333±15 and 463±43 ng μl-1 of RNA with RNA integrity (RIN) numbers of 8.6 and 9.05 were obtained from roots under cadmium stress and control conditions using the described optimized method, respectively.ConclusionIn general, the results of this study showed that the modified RNX-Plus method is convenient, fast, and effective for the isolation of total RNA from borage root and leaf tissues that contain different levels of polysaccharides, polyphenols, and secondary metabolites, and no solution is needed to be prepared before, except for ethanol and Lithium chloride. Since the RNA extracted from this procedure was successfully used for cDNA library construction, RT-PCR, and RNA sequencing, it can be considered as a simple and efficient method for the isolation of RNA from medicinal plants.

Keywords

• borago officinalis
• cdna library
• heavy metal
• rna-seq
• rna integrity