The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.
Part 1: Extraction of Total RNA using TRIzol Reagent
When 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 Kit
The Qiagen RNEasy Mini Kit2 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.
Part 3: cDNA Synthesis using the Invitrogen SuperScript First-Strand System
RNA is easily prone to cleavage by RNAses leading to degradation. As a result many gene expression protocols have been developed to use a more stable cDNA product that has been directly synthesized from the RNA. Here we detail synthesis of first-strand cDNA using the Invitrogen SuperScript First-Strand Synthesis System3. Each cDNA synthesis reaction can accommodate up to 5 μg of RNA.
Part 4: cDNA Isolation and Precipitation
Representative Results
After RNA cleanup, approximately 15 μg of high quality total RNA can be expected from 50 zebrafish embryos. To assess total RNA quality, a spectrophotometer, gel electrophoresis, and an Agilent 2100 Bioanalyzer can be used. A NanoDrop ND-1000 spectrophotometer can be used to assess the absorbance at 260 nm in comparison to the absorbance at 280 nm (Figure 1). The A260/A280 ratio can reveal the presence of contaminants and give evidence of possible degradation. An A260/A280 ratio of 1.9-2.0 is considered acceptable. In addition, it is highly recommended that an RNA denaturation gel be run to assess RNA integrity. RNA strands will separate on basis of their size, with smaller strands migrating further down the gel. A non-degraded sample will appear as a smear with two strong bands. The smear is a result of a population of different sized mRNA strands and the bands correspond to 28S and 18S rRNA. The 28S band should be about twice as intense as the 18S band (Figure 2). Alternatively, total RNA integrity can be assessed using an Agilent 2100 bioanalyzer. Two distinct peaks representing the 28S and 18S rRNA is expected (Figure 3). The 28S peak should be larger than the 18S peak with the area under the peaks approximately 2:1, respectively. Two distinct peaks will not be observed in degraded samples. RNA samples exhibiting degradation should not be carried through subsequent experimental procedures.
Spectrophotometric analysis can be used to assess the quality of the cDNA product (Figure 4). The A260/A280 should be around 1.8. cDNA reactions with an input of 5 μg of total RNA routinely yield 1-2 μg of cDNA product in our laboratory.
Figure 1. A NanoDrop spectrum of RNA sample. After RNA cleanup, RNA quantity and quality can be assessed using a NanoDrop ND-1000 spectrophotometer. Approximately 15 μg of high quality total RNA is routinely yielded from 50 zebrafish embryos with an A260/A280 ratio around 1.9-2.0.
Figure 2. An RNA denaturation gel. To assess the integrity of the total RNA isolation, an RNA denaturation gel can be ran. A smear with two bright bands corresponding to 28S and 18S rRNA is expected. The 28S band should be approximately twice as intense as the 18S band.
Figure 3. A trace of an RNA sample from a bioanalyzer. To assess the integrity of the total RNA isolation, samples may also be analyzed on an Agilent 2100 bioanalyzer. Two sharp peaks, corresponding to the 28S and 18S rRNA, should be visible. The 28S peak should be larger than the 18S peak with the area under the peaks approximately 2:1, respectively.
Figure 4. A NanoDrop spectrum of a cDNA sample. Spectrophotometric analysis with a NanoDrop ND-1000 can be used to assess the quantity and quality of the cDNA product. The A260/A280 ratio should be around 1.8. cDNA reactions with an input of 5 ug of total RNA routinely yield 1-2 ug of cDNA in our laboratory.
Table 1. Components to add for RNA/primer mixtures at step 2 of cDNA synthesis.
Component | Volume |
Total RNA (up to 5 μg) | 10 μl |
10 mM dNTP mix | 1 μl |
Oligo(dT)12-18 (0.5 μg/ μl) | 1 μl |
DEPC-treated water | n μl |
Total volume | 10 μl |
Table 2. Reaction mixture for step 5 of cDNA synthesis. Volumes listed are per reaction. Increase volumes of each component for the total number of reactions.
Component | Volume |
10X RT Buffer | 2 μl |
25 mM MgCl2 | 4 μl |
0.1 mM DTT | 2 μl |
RNAseOUT | 1 μl |
The fragility of the RNA molecule is the most critical consideration that should be remembered throughout this protocol. Sterile equipment that is RNAse-free should always be used in an RNAse-free zone of the laboratory. It is helpful to reserve a part of the laboratory to be used for RNA procedures only. This area should be frequently sprayed with an RNAse removing product, such as RNAse Away. The RNA samples should always be handled with gloves and kept on ice to prevent degradation.
This protocol took advantage of commercially available reagents and kits. Alternatively, there are numerous additional reagents and RNA isolation kits available on the market for total RNA isolations. The reagents and kits included in this protocol have worked well and routinely yielded high quality RNA in our laboratory.
As an alternative, double stranded cDNA synthesis can be performed, instead of the first strand synthesis demonstrated in this protocol. Double stranded cDNA is sometimes desired as it provides increased stability. In addition, some protocols require double stranded cDNA products as an input. However, if the samples are going to be kept in house for downstream applications, first strand synthesis is sufficient and is much more economical.
The authors have nothing to disclose.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Glycogen (5 mg/ml) | Ambion | 9510 | ||
NanoDrop ND-100 | Thermo Scientific | ND-1000 | ||
Pellet Pestles with Microfuge Tube | VWR | KT749520-0090 | ||
Phase Lock Gel Light, 1.5 ml | Eppendorf | 2302800 | ||
Phenol, Tris-saturated | Roche Diagnostics | 3117944001 | ||
RNAse Away | VWR | 17810-491 | ||
RNAse-Free DNase Set | Qiagen | 79254 | ||
RNEasy Mini Kit | Qiagen | 74104 | ||
SuperScript® First-Strand Synthesis System for RT-PCR | Invitrogen | 11904-018 | ||
TRIzol | Invitrogen | 15596-026 |