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Evaluation of two independent protocols for the extraction of DNA and RNA from different tissues of sea cucumber Isostichopus badionotus.
Puch-Hau C
,
Sánchez-Tapia IA
,
Patiño-Suárez V
,
Olvera-Novoa MA
,
Gullian Klanian M
,
Quintanilla-Mena M
,
Zapata-Pérez O
.
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Isostichopus badionotus is a sea cucumber species of great ecological and economic relevance for Mexico and Central American and Caribbean countries; however, the protocols for the extraction of the nucleic acids have not yet been published. In this study, we describe the first protocols to obtain DNA and RNA from different tissues of I. badionotus, which include the respiratory tree, gonad, longitudinal muscle bands, anterior intestine and cloaca. The extraction of high-quality DNA was performed using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) with minor modifications in different points of the protocol. Concerning the RNA, the method of TRIzol was used. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when the separation of cytoplasmic RNA from nuclear RNA is impractical. The methodologies used in this study allowed us to obtain DNA and RNA of high quality and integrity in the different tissues of I. badionotus, which will be the basis for future genomic and transcriptomic studies. •The successful extraction of DNA and RNA was achieved in the different tissues of I. badionotus.•The concentrations of DNA and RNA obtained were adequate for a diversity of analyses at a molecular level.
Fig. 1. Specimens of I. badionotus dissected for the extraction of DNA and RNA. (a) Color variants, (b) Respiratory Tree, (c) Gonad, (d) Cloaca and Longitudinal Muscle Bands (LMB) and (e) Anterior Intestine (AI).
Fig. 2. Nucleic acids visualized in an agarose gel 2%, extracted from different tissues of four sea cucumbers. (a) Genomic DNA and (b) total RNA. LRT: Left Respiratory Tree, RRT: Right Respiratory Tree, G: Gonad, M; Muscle, AI: Anterior Intestine and C: Cloaca. The numbers correspond to the dissected organisms. MM: molecular mass marker (1â¯kb).
Fig. 3. Amplified fragment of Ë150â¯bp corresponding to 18S rRNA gene visualized in an agarose gel 2%. (a) DNA and (b) cDNA samples. LRT: Left Respiratory Tree, RRT: Right Respiratory Tree, M; Muscle G: Gonad, AI: Anterior Intestine and C: Cloaca. + positive control, â negative control and MM: molecular mass marker (1â¯kb).
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