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. 2020 Oct;10(10):433.
doi: 10.1007/s13205-020-02424-w. Epub 2020 Sep 16.

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

Suman Dutta  1 , Vignesh Muthusamy  1 , Rashmi Chhabra  1 , Rajkumar U Zunjare  1 , Firoz Hossain  1
Affiliations

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

Suman Dutta et al. 3 Biotech. 2020 Oct.

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 (A 260/280) 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 A 260/280 values of isolated RNA were 1.984 ± 0.030. DNase treatment further improved the A 260/280 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: Dry condition; Leaf; RNA; Seed; Starch; Storage.

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Conflict of interest statement

Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest. The authors declare that they have no conflict of interests.

Figures

Fig. 1
Fig. 1
Isolation of RNA from seed and leaf tissues. Lane no. 1 to 5: seed sample at monthly intervals (up to 4 months); Lane no. 6: fresh leaf sample
Fig. 2
Fig. 2
Removal of DNA contamination in the samples after DNase treatment. Lane no. 1–5: seed sample at monthly intervals (up to 4 months); Lane no. 6: fresh leaf sample
Fig. 3
Fig. 3
Average RNA yield (ng/µl) form seeds and leaf sample. DAS days after storage for seeds; error bar represents standard deviation around mean
Fig. 4
Fig. 4
Amplification of Adh1 gene in the samples. Lane no. 1–5: seed sample at monthly intervals (up to 4 months); Lane no. 6: fresh leaf sample
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