Double lysis: an integrative time-saving method yielding high-quality RNA from strawberry

Abstract

Background: The isolation of high-quality RNA from strawberry leaves and fruits is notoriously cumbersome. Both tissues are extremely rich in active secondary metabolites, as phenolics and pigments that inevitably perturb the isolation of RNA. Many protocols have been developed to address this problem. However, they are either costly, or time-consuming, in particular for high number of many plant samples. We describe here a new method with an easy-to-handle approach to obtain high-quality RNA from strawberry leaves and fruits. The method, referred to as double lysis, uses a novel combination of CTAB and Trizol protocols.

Results: Compared to conventional Trizol-dependent protocols, either used individually, or in a commercial spin-column kit, the new method yields RNA at lower costs, but of significantly improved quality. The RNA obtained by double lysis was very pure as indicated by 260/280 ratios of 2.06 (leaves) and 2.07 (fruits), while 260/230 ratio was 2.07 and 1.75, respectively. Also visually, RNA sediments from double lysis showed a white color, indicative of efficient elimination of polyphenolics and pigments. In contrast, traditional Trizol method produced reddish precipitates. The purity of RNA isolated by double lysis enabled successful removal of genomic DNA and, thus, allowed for more efficient cDNA synthesis and RT-PCR. In addition, the suggested method is able to yield RNA with fully preserved integrity from strawberry leaves, fruits in addition to petals and roots.

Conclusion: Double lysis is a new RNA isolation protocol developed through the integrative coupling of CTAB and Trizol reagents. The method is cost-effective, robust, time-saving, and can cope even with recalcitrant plant tissues with high contents of phenolics and pigments, such as strawberry leaves and fruits.

Keywords: Double lysis; Fragaria × ananassa; Fruits; Leaves; RNA; Strawberry.

Fig. 1

Variation in the appearance of RNA sediments isolated from strawberry leaves, a by standard Trizol-based extraction and b as compared to the double-lysis method

Fig. 2

Integrity of RNA isolated from strawberry leaves a and fruits b assayed by electrophoretic separation using different extraction protocols: double-lysis method (left), conventional Trizol extraction and extraction by the Direct-zol kit. c Agarose gel electrophoresis showing the migration of RT-PCR product of a fragment of FaGAPDH2 spanning an intron. The expected size of the product is 200 bp in case of a genomic DNA template, and 91 bp in case of a cDNA template, either Direct-zol or Trizol samples are from leaves RNA. Templates obtained from RNA extracted by the double-lysis method from fruits (Fr) and leaves (Lv) are compared to templates from standard Trizol extraction or the spin-column based Direct-zol kit. Gene Ruler^TM 100 bp plus DNA ladder as M

Fig. 3

Total RNA isolation from leaves, fruits, roots, and petals of strawberry plants using double-lysis method. M Gene Ruler ^TM 100 bp DNA marker

Fig. 4

Purity of RNA isolated from strawberry leaves (left) and fruits (right) assayed spectrophotometrically. aA₂₆₀/A₂₈₀ ratio bA₂₆₀/A₂₃₀ ratio. Values represent means ± SE of three independent biological replications. Means with the same letters are not significantly different according to Tukey’s honest significant (HSD) test (p ≤ 0.05)

Fig. 5

Schematic flow diagram for isolating RNA from strawberry leaves, fruits, petals, and roots using double-lysis method