مقایسه روش‏های استخراج rna به منظور بهینه ‏سازی استخراج rna کل از گیاه گاوزبان اروپایی تحت تنش کادمیوم

کیفیت و کمیت مناسب rna برای انجام موفقیت‌آمیز مطالعات پروفایل بیان ژن مانند توالی ‏یابی rna و ریزآرایه ‏ها مسئله ضروری است. جداسازی rna از گیاهان دارویی مانند borago officinalis که حاوی مقادیر بالایی از پلی‏ فنل ‏ها و پلی‏ ساکاریدها هستند، چالش بزرگی است. همچنین استخراج اسیدهای نوکلئیک از بافت‌های گیاهی که در معرض شرایط تنش‌زای ناشی از سمیت فلزات سنگین ازجمله کادمیوم قرار دارند به دلیل افزایش تجمع گونه‌های فعال اکسیژن و متابولیت‌های‌ثانویه دشوار است. در مطالعه حاضر پنج روش استخراج rna شامل روش‏های مبتنی بر فنل-کلروفرم، ctab، sds، محلول استخراج rnx-plus و rnx-plus تغییریافته برای استخراج rna از ریشه و برگ گاوزبان اروپایی در معرض تنش کادمیوم مقایسه شد. پس از بررسی روش‏های استخراج، یک دستورالعمل سریع، ساده و کارآمد مبتنی بر روش استخراج rnx-plus تغییریافته ارائه شد که بر محدودیت‌های ناشی از کیفیت پایین و عملکرد کم rna جداشده از این گیاه غلبه می‌کند. مقادیر نسبت‌های a260/a280 و a260/a230 برای rna حاصل از روش rnx-plus تغییریافته به ترتیب 2.1 و 2.07 بود که این نتایج خلوص بالای آن را نشان می‏دهد. عوامل موثری که منجر به حذف ناخالصی‏ ها در این روش تغییریافته شدند عبارت‌اند از: افزایش نسبت بافر استخراج به نمونه گیاهی پودر شده، استفاده از حجم بهینه کلروفرم، افزایش زمان ته ‏نشینی در دمای 20- درجه سانتی‌گراد، شستشو rna با لیتیوم کلراید و شستشو مجدد با اتانول. همچنین غلظت 15± 333 و 43± 463 نانوگرم در میکرولیتر rna با عدد یکپارچگی rna  برابر با 8.6 و 9.05 به ترتیب از ریشه‏های تحت تنش کادمیوم و شاهد به دست آمد. ازآنجایی‌که rna حاصل استانداردهای کنترل کیفیت را برای ساخت کتابخانه cdna، rt-pcr و توالی‏ یابی rna گذرانده است، می‏ توان آن را به‌عنوان روشی ساده و کارآمد برای استخراج rna از این گیاه دارویی در نظر گرفت.

comparison of rna extraction methods in order to optimize total rna extraction from borage tissues under cadmium stress

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.