An optimized isolation protocol yields high-quality RNA from cassava tissues (Manihot esculenta Crantz)

Abstract

We developed and modified a precise, rapid, and reproducible protocol isolating high-quality RNA from tissues of multiple varieties of cassava plants (Manihot esculenta Crantz). The resulting method is suitable for use in mini, midi, and maxi preparations and rapidly achieves high total RNA yields (170-600 μg·g^-1) using low-cost chemicals and consumables and with minimal contamination from polysaccharides, polyphenols, proteins, and other secondary metabolites. In particular, A₂₆₀ : A₂₈₀ ratios were > 2.0 for RNA from various tissues, and all of the present RNA samples yielded ribosomal integrity number values of greater than six. The resulting high purity and quality of isolated RNA will facilitate downstream applications (quantitative reverse transcriptase-polymerase chain reaction or RNA sequencing) in cassava molecular breeding.

Keywords: RNA isolation; RNA sequencing; cassava; qRT‐PCR.

Figure 1

Electropherograms of total RNA from cassava obtained using our method showing 18S and 25S rRNA regions with RNA concentrations and RIN values; (A) to (C) correspond with RNA from leaves and storage roots, and (A) and (B) correspond with RNA from different stages of plant development (young and mature leaves). RNA were visualized in denaturing agarose gels stained with SYBR safe. RNA were analyzed using Agilent RNA 6000 Nano Assays in a 2100 Bioanalyzer (Agilent Technologies) and were then used for RNA sequencing.

Figure 2

Electropherogram of sequencing libraries; the graph shows length distribution curves of sequencing libraries obtained using a low‐cost library construction protocol 18. Curves were generated on a 2100 Bioanalyzer using a DNA 1000 chip (Agilent Technologies). The photograph was provided by Maria Irigoyen and Linda Walling, University of California, Riverside.

Figure 3

Conventional PCR with G3pdh primers was performed using cDNA that was reverse‐transcribed from total RNA after extraction using the present protocol. Tissue samples were taken from HMC‐1 (A) and Esparrago (ESP; (B) genotypes (Manihot esculenta) that were grown under long‐day (LD) conditions for indicated times; M: 1 Kb ladder; 85: HMC‐1, 0 h LD; 86: HMC‐1, 4 h LD; 87: HMC‐1, 8 h LD; 88: HMC‐1, 12 h LD; 89: HMC‐1, 16 h LD; 90: HMC‐1, 20 h LD; 91: HMC‐1, 24 h LD; 197: ESP, 0 h LD; 198: ESP, 4 h LD; 199: ESP, 8 h LD; 200: ESP, 12 h LD; 201: ESP, 16 h LD; 202: ESP, 20 h LD; 203: ESP, 24 h LD; NTC: non‐template negative control (water template), and gDNA: cassava genomic DNA.

Figure 4

Conventional PCR using primers for G3pdh and cDNA from total RNA that was extracted from various tissues of the ESP genotype grown in pots and in vitro at 15 °C and 30 °C under LD conditions for 16 h inside growth chambers. Biological repeat 1: left panel; M, 1 Kb ladder; TL48, buds and young leaves from potted plants at 15 °C; TL54, stems from potted plants grown at 15 °C; right panel, NTC, negative control, water template; gDNA, cassava genomic DNA; IL126, roots from in vitro samples grown at 30 °C; IL138, leaves from in vitro samples grown at 30 °C; biological repeat 2: IL141, stems from in vitro samples grown at 30 °C. See Fig. S2 for complete PCR analyses of total RNA from potted and in vitro samples.

Figure 5

Quality analyses of cDNA from total RNA that was extracted using the present modified protocol; leaf samples were taken from the cassava genotypes HMC‐1 and ESP at indicated time points, and qRT‐PCR analyses were performed using primers for 18S. (A) Amplification plot and melting curves for HMC‐1 genotype; (B) amplification plot and melting curve for ESP genotype. For details of all curves, see Fig. 3 legend.

Figure 6

Amplification plots and melting curves for amplicons generated using cDNA from tissues that were taken from esparrago (ESP) genotype grown in pots and in vitro; qRT‐PCR analyses were performed using primers for 18S. M, 1 Kb ladder; biological repeat 1: TL48, buds and young leaves from pot samples grown at 15 °C; TL54, stems from potted plants grown at 15 °C; biological repeat 2; TL120, buds and young leaves from potted plants grown at 30 °C; TL126, stems from potted plants grown at 30 °C; IL48, leaves from in vitro plants grown at 15 °C; IL51, stems from in vitro plants grown at 15 °C; IL54, roots from in vitro plants grown at 15 °C; IL120, leaves from in vitro plants grown at 30 °C; IL123, stems from in vitro plants grown at 30 °C; IL126, roots from in vitro plants grown at 30 °C; NTC, negative control, water template.