RNA isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis.
Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.
Peterson
Samuel M
SM
School of Health Sciences, Purdue University.
Freeman
Jennifer L
JL
D000818:Animals; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000235:genetics* / Q000302:isolation & purification; D020869:Gene Expression Profiling; D012313:RNA / Q000302:isolation & purification*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
D018076:DNA, Complementary; D012313:RNA