Sasi Shina, Krishnan Saranya, Kodackattumannil Preshobha, Shamisi Aysha Al, Aldarmaki Maitha, Lekshmi Geetha, Kottackal Martin, Amiri Khaled M A
Khalifa Center for Genetic Engineering and Biotechnology, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates.
Department of Biology, College of Science, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates.
Plant Methods. 2023 Aug 11;19(1):84. doi: 10.1186/s13007-023-01063-5.
High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications.
Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A/A and A/A ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 C. Pre-warming of the buffer for 5-10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol's broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds.
The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.
高纯度RNA是植物物种下游分子分析的基本要求，尤其是基因对各种生物和非生物刺激的差异表达分析。但是，从富含多糖和多酚的植物中提取高质量RNA通常很困难，而且它们的存在通常会干扰下游应用。本研究的目的是优化从多种植物物种/组织中提取高质量RNA的方法，以用于下游分子应用。
使用市售RNA提取试剂盒和常规十六烷基三甲基溴化铵（CTAB）方法从灰叶豆、银叶树和海枣中提取RNA，均未得到高质量的无DNA RNA。在筛选了九种不同方法后，优化出了一种可靠的从这些难提取树木的成熟叶片中提取高质量RNA的方案。一种无需DNase I和蛋白酶K处理的改良方法，即先用CTAB法提取，再用TRIzol提取，得到了A260/A280和A260/A230比值均>2.0的高质量无DNA RNA。对于最难提取的银叶树，通过避免将研磨后的组织在65℃缓冲液中加热孵育成功提取到了RNA。将缓冲液预热5 - 10分钟就足以提取到高质量的RNA。提取的RNA样品的RNA完整性数值在7到9.1之间，凝胶电泳显示出完整的28S和18S RNA条带。从灰叶豆RNA构建的cDNA文库用于下游应用。使用灰叶豆RNA的cDNA进行的实时定量PCR分析证实了其质量。从2021年交替月份（1月至12月，涵盖冬季、春季、秋季以及非常干燥炎热的夏季）采集的沙漠生长的灰叶豆（>20年树龄）样本中提取高质量RNA，证明了该方案的有效性。通过从36种难提取的植物物种（包括根、花、花器官、果实和种子等组织）中提取高质量RNA，进一步验证了该方案的广泛适用性。
改良的无需DNase I和蛋白酶K处理的方案能够从属于32个被子植物科的总共39种难提取植物物种中提取无DNA、高质量、完整的RNA，无论组织类型和生长季节如何，都有助于从双子叶植物和单子叶植物中提取高质量RNA。
