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Two-step method for isolation of high-quality RNA from stored seeds of maize rich in starch

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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.

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Acknowledgements

The first author is thankful to Indian Council of Agricultural Research for the Junior Research Fellowship to pursue his master’s degree programme.

Funding

Financial support from the ICAR funded Consortia Research Platform on ‘Biofortification of Selected Crops for Nutritional Security-Maize Component’ [IARI Project Code No.: 12-131] is thankfully acknowledged.

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Authors and Affiliations

  1. Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

    Suman Dutta, Vignesh Muthusamy, Rashmi Chhabra, Rajkumar U. Zunjare & Firoz Hossain

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  1. Suman Dutta
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  2. Vignesh Muthusamy
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  3. Rashmi Chhabra
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  4. Rajkumar U. Zunjare
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  5. Firoz Hossain
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Contributions

Conduct of the experiment: SD; RNA isolation from seeds: MV; RNA isolation from leaves: RC; field experiment and sample preparation: RUZ; drafting of the manuscript: SD, MV and FH; design of experiment: MV and FH.

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Correspondence to Vignesh Muthusamy.

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Dutta, S., Muthusamy, V., Chhabra, R. et al. Two-step method for isolation of high-quality RNA from stored seeds of maize rich in starch. 3 Biotech 10, 433 (2020). https://doi.org/10.1007/s13205-020-02424-w

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