Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for Next Generation Sequencing

Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high‐quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash‐frozen and stored at −80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at −80 °C, RNAlater and storage at −20 °C, preservation in TRIzol reagent and storage at −80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips

Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for Next Generation Sequencing

6 páginas, 3 figuras, 1 tablaTranscriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at −80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at −80 °C, RNAlater and storage at −20 °C, preservation in TRIzol reagent and storage at −80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips.This research was supported by ‘Juan de la Cierva’ contracts (Spanish Government) to R.P-P and A.R. Funding was provided by the Spanish Government projects CTM2010-22218 (BENTHOMICS) and CTM2010-17755 (SOLID).Peer reviewe