Two-step method for isolation of high-quality RNA from stored seeds of maize rich in starch
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Two‑step method for isolation of high‑quality RNA from stored seeds of maize rich in starch Suman Dutta1 · Vignesh Muthusamy1 · Rashmi Chhabra1 · Rajkumar U. Zunjare1 · Firoz Hossain1 Received: 21 April 2020 / Accepted: 2 September 2020 © King Abdulaziz City for Science and Technology 2020
Abstract A modified SDS–Trizol method was optimized for isolation of total RNA from the stored maize seeds at regular interval of one month for 4 months. Use of SDS extraction buffer before the use of Trizol reduced the co-precipitation problem associated with high carbohydrate content in the seed. Recorded mean RNA yield from seeds across the storage intervals was 978.6 ± 65.46 ng/µl. Average spectrophotometric values (A260/280) of isolated RNA varied from 1.974 ± 0.033 to 1.998 ± 0.022. Attempts to isolate RNA from green leaves using Trizol method also ensured comparable quality and quantity of the isolated RNA. RNA yield from fresh leaves was recorded 1008.2 ± 77.088 ng/µl which is slightly higher than the mean RNA yield from seeds across months. Observed mean A260/280 values of isolated RNA were 1.984 ± 0.030. DNase treatment further improved the A260/280 ratio in both seeds (2.003 ± 0.006) and leaves (2.012 ± 0.037). High quality and quantity along with integrity of the isolated RNA was ensured through downstream analysis after RNA extraction such as first-strand cDNA synthesis and normal PCR. Extraction of RNA from the stored seeds using modified SDS-based Trizol method and from fresh leaves using Trizol method opened new possibility of understanding role of key genes involving developmental steps especially in the stored seeds. Keywords RNA · Seed · Leaf · Dry condition · Starch · Storage
Introduction Maize serves as a staple food, feed and industrial by-products worldwide (Prasanna et al. 2020). Understanding the genetic makeup is key to the success of development of cultivars as per the need (Wimalanathan et al. 2018). In the post-genomic era, considerable successes have been achieved to structurally dissect the whole genome of crops and functional annotation of target genes (Bouchez and Hofte 1998). However, spatial and temporal expressions of the genes in various parts of the tissue made it more difficult to understand the whole genetic make-up of crop species (Koltunow et al. 1990; Dutta et al. 2019a). In this context, isolation of RNA becomes an initial step to understand the functionality of genome (Tan and Yiap 2009). RNA is one of the key elements involving in entire machinery of the living system (Chomczynski and Sacchi * Vignesh Muthusamy [email protected] 1
Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India
1987). Therefore, isolation of high-quality RNA from different part of the plant tissues becomes an important step to understand the fundamental biological processes. However, quality of the isolated RNA varies among various tissues due to varying chemical composition (Pereira et al. 2017; Liu et al. 2018). In the crop like cereals,
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