RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Abstract

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. Protocol Part 1: Extraction of Total RNA using TRIzol ReagentWhen working with RNA it is very important to work in an environment that is free of RNAses. Simple precautions such as having reserved pipettes for use only with RNA procedures and spraying the work area with a decontaminant reagent (e.g., RNAse Away) before beginning the procedure are very helpful. Part 2: RNA Cleanup using the Qiagen RNEasy Mini KitThe Qiagen RNEasy Mini Kit 2 is useful in removing contaminants and impurities from an RNA sample isolated from other methods. For this procedure, β-mercaptoethanol, ethanol, and nuclease-free water are needed in addition to the components included in the kit. An optional DNAse treatment is recommended during this procedure.

A Genome-wide Screen Identifies PAPP-AA-Mediated IGFR Signaling as a Novel Regulator of Habituation Learning

“…To confirm candidate nonsense mutations in pappaa and pcxa , cDNA was prepared from total mRNA extraction from 5 dpf larvae as previously described (Peterson and Freeman, 2009). pappaa and pcxa cDNA were amplified with primers (Supplemental Table 1) designed against pappaa and pcxa reference sequence (Ensembl) with the following RT-PCR conditions: 94°C for 3 min and then 35 cycles of 94°C for 30 sec, 57°C for 1 min, and 70°C for 1 min.…”

Novel cardiovascular gene functions revealed via systematic phenotype prediction in zebrafish

“…Overexpression of tmem88a RNA was isolated from 48 hpf wild-type embryos and used to synthesize cDNA as previously described (Peterson and Freeman, 2009). tmem88a-specific primers containing the MultiSite Gateway attB1 and attB2 recognition sites were then used to amplify tmem88a from cDNA by PCR.…”

“…Pacing at a constant rate of 80 beats per minute was used for calcium mapping to eliminate heart rate effects. For all comparisons, regions of interest were determined and compared between morpholino-injected embryos and controls from at least two separate mate pairings using a two-sided Student's t-test.Overexpression of tmem88a RNA was isolated from 48 hpf wild-type embryos and used to synthesize cDNA as previously described (Peterson and Freeman, 2009). tmem88a-specific primers containing the MultiSite Gateway attB1 and attB2 recognition sites were then used to amplify tmem88a from cDNA by PCR.…”

Water Contaminated With the Herbicide Atrazine: Alteration of Cancer Associated Tumor Protein D52-like 1 (tpd52l1) Gene Using the Zebrafish Model

“…The homogenized samples were then flash frozen in liquid nitrogen and stored at -80ºC until RNA isolation. RNA isolation was done using the RNEasy kit (Qiagen), and cDNA was synthesized using the First Strand Synthesis Kit (Life Technologies) which follows established protocols (Peterson and Freeman, 2009). Gene expression was then quantified using qPCR for the target gene: tpd52l1.…”