For miRNA analysis, zebrafish blood was obtained by RO technique and pooled as described in results. Serum was separated by centrifugation and miRNA was extracted using a miRNeasy kit (Qiagen, Venlo, Netherlands) after which the small RNA fraction was purified using a MinElute kit (Qiagen, Venlo, Netherlands).8 (link) MiRNAs were measured using Taqman-based quantitative PCR.
The small RNA elute was reverse transcribed using specific stem-loop reverse-transcription RT primers (Applied Biosystems, Foster City, CA, USA) for each target miRNA species, following the manufacturer’s instructions. In the reverse transcription reaction, 2 μl of RNA was used to produce the complementary DNA (cDNA) template in a total volume of 15 μl. Then, 1.33 μl of cDNA was used in the PCR mixture with specific PCR primers (Applied Biosystems, Foster City, CA) in a total volume of 20 μl. Levels of miRNA were measured using the Light Cycler 480 (Roche, Basel, Switzerland). All samples were assayed in duplicate. MiRNA levels were normalised to levels of let-7d which has been reported to be an appropriate internal normaliser in humans.20 (link) At the time of writing the optimum normaliser for zebrafish was not described.
The small RNA elute was reverse transcribed using specific stem-loop reverse-transcription RT primers (Applied Biosystems, Foster City, CA, USA) for each target miRNA species, following the manufacturer’s instructions. In the reverse transcription reaction, 2 μl of RNA was used to produce the complementary DNA (cDNA) template in a total volume of 15 μl. Then, 1.33 μl of cDNA was used in the PCR mixture with specific PCR primers (Applied Biosystems, Foster City, CA) in a total volume of 20 μl. Levels of miRNA were measured using the Light Cycler 480 (Roche, Basel, Switzerland). All samples were assayed in duplicate. MiRNA levels were normalised to levels of let-7d which has been reported to be an appropriate internal normaliser in humans.20 (link) At the time of writing the optimum normaliser for zebrafish was not described.