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Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next‐generation sequencing

2011; Wiley; Volume: 12; Issue: 2 Linguagem: Inglês

10.1111/j.1755-0998.2011.03097.x

1755-0998

Ana Riesgo, Alicia Pérez-Porro, Susanna Carmona, Sally P. Leys, Gonzalo Giribet,

Marine Ecology and Invasive Species

Abstract Transcriptome sequencing with next‐generation sequencing technologies has the potential for addressing many long‐standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high‐quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN 2 ) and (ii) immersion in RNA later , for the recovery of high‐quality mRNA from sponge tissues. We also tested whether the long‐term storage of samples at −80 °C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN 2 outperforming RNA later ), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN 2 ‐preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNA later lost efficiency in storage times longer than 2 months compared with flash‐frozen samples. In summary, we find that for long‐term preservation of samples, flash freezing is the preferred method. If LN 2 is not available, RNA later can be used, but mRNA extraction during the first month of storage is advised.