Easy RNA purification with integrated DNA-removal step
Karen Dolter Jeff Braman
The StrataPrep total RNA miniprep kit efficiently isolates high-quality total RNA from 10 5 to 10 7 cultured cells or 10 to 40 mg of tissue and includes an on-column DNA-removal step. Additionally, multiple samples can be processed simultaneously because of the easy-to-follow technique. The RNA isolation is performed within microspin cups, which eliminates the need for organic extraction or ethanol precipitation. The resulting total RNA is suitable for a wide variety of demanding molecular biology applications.
High-quality RNA is necessary for techniques such as cDNA synthesis, RT-PCR amplification, RNase protection assay, primer extension analysis, and Northern blotting. Stratagenes StrataPrep total RNA miniprep method takes advantage of the efficient denaturing properties of guanidine thiocyanate for cell lysis. The protein-denaturing ability of this chaotropic salt facilitates the isolation of intact RNA from tissue rich in ribonucleases. The method originally described1 has been simplified by using a silica-based fiber matrix in a microspin-cup format (Figure 1). The RNA is immobilized on the fiber matrix, allowing contaminant removal while avoiding organic extractions. In addition, no ethanol precipitation of the RNA is necessary. By treating the RNA sample directly on the fiber matrix with DNase, DNA contamination is reduced to undetectable levels. Once the purified RNA is eluted from the fiber matrix in a small volume of buffer or water, it is ready to use.
We showed that the kit is capable of high performance using a wide range of starting materials, both tissues and cells. We then assessed the quality of the isolated RNA by absorbance measurements, gel electrophoresis, RT-PCR, and molecular beacon real-time RT-PCR.
RNA Yield and Purity from Varying Amounts of Rat Liver
RNA was isolated from the specified amounts of tissue using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.
Amount of rat liver
Average yield (g)
Table 1 includes yield and purity data for RNA isolated from varying amounts of rat liver, using the kit. The RNA yields were good, averaging 14 to 122 g for liver quantities between 5 and 40 mg; individual yields reached as high as 141 g. The purity, as determined by the A260/A280 ratio, was 1.9 or greater (Table 1).
Isolation of RNA from Different Cell Lines
RNA was isolated from 2 x 106 cells using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.
Average yield (mg/2 x 106 cells)
Table 2 and Table 3 show yield and purity of RNA isolated from a variety of cells and tissues. The RNA exhibited A260/A280 ratios of greater than 1.9 for each sample, indicating high purity of the RNA. Approximately 1 g of representative RNA samples was electrophoresed on formaldehyde-agarose gels to assess the integrity of the RNA. Figure 2 shows intact 28S and 18S ribosomal RNA bands for each sample.
Isolation of RNA from Various Mouse Tissues
RNA was isolated from different amounts of mouse tissues using the StrataPrep total RNA miniprep kit. Yield and purity were determined by measuring absorbance in 10 mM Tris, pH 7.5.
Average yield (mg/mg tissue)
Number of isolations
With the RT-PCR technique, small quantities of genomic DNA contamination can be detected in RNA samples. This method was used to demonstrate the effective removal of DNA using the StrataPrep total RNA miniprep kit (Figure 3). Primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences (present in approximately 200 copies in mouse and rat genomes2) were used, providing an especially sensitive assay for the presence of DNA. PCR amplification of GAPDH sequences from cDNA shows the expected 0.2-kb fragment (Figure 3: Lanes 1, 3, 5, and 7). When the DNase treatment step is omitted from the protocol and reverse transcriptase is omitted from the cDNA synthesis reaction, the presence of DNA is detected in the PCR (Figure 3: Lanes 4 and 8). However, when the StrataPrep total RNA miniprep protocol is carried out according to the kits instructions, no DNA contamination is detected (Figure 3: Lanes 2 and 6).
RNA isolated with the StrataPrep total RNA miniprep kit was used to synthesize cDNA, which served as a template for amplification using Stratagenes RT-PCR Primers for Human Gene Transcripts of Varying Abundance Levels,3 according to the instructions provided. The presence of PCR products for high- (-actin), medium- (g-actin) and low- (protein phosphatase 1, ADP ribosylation factors 1 and 3, and ornithine decarboxylase) abundance mRNA shows that a wide spectrum of RNA molecules is present in the sample (Figure 4).
Molecular beacon technology provides an elegant and efficient method for PCR and RT-PCR that does not require gel electrophoresis of the products and permits accurate quantitation.4 The Mx4000 molecular beacon expression analysis kit for mouse -actin5 was used for real-time RT-PCR analysis of RNA isolated from NIH/3T3 cells with the StrataPrep total RNA miniprep kit. RNA is readily detected from a sample as small as 1 pg in a target titration of 1 100,000 pg of total RNA (Figure 5). These results demonstrate that the RNA isolated using the StrataPrep kit is of the high quality required for real-time RT-PCR.
Most spin-cup-based total RNA isolation kits from other manufacturers do not include a DNase digestion step in the standard protocol. When these kits are used for samples that require DNA removal, DNase treatment must be performed after isolating the RNA, which is then followed by repurifying the RNA to remove the DNase. Extra materials are not provided in these kits to perform these steps. In some kits that include DNase digestion within the protocol, only very small quantities of tissue and/or cells can be used. Other RNA isolation kits that provide DNase require extra steps, such as heating and a long centrifugation or repurification of the RNA.
The StrataPrep total RNA miniprep kit is a quick and convenient method for isolating pure total RNA with high yields and no evidence of DNA contamination from a variety of cells and tissue. Use the purified RNA in cDNA synthesis, RT-PCR amplification, RNase protection assay, primer extension analysis, Northern blotting, and other applications. Additionally, follow this procedure to purify RNA after enzyme reactions (to remove RNA polymerase, DNase, etc.).
To isolate RNA, tissue or cultured cells are homogenized in lysis buffer, then the sample is passed through a prefilter by centrifugation. The prefilter removes particulates and much of the DNA contamination. The clarified homogenate is mixed with ethanol and applied to the fiber matrix within the microspin cup for binding of the RNA. The RNA is washed, then the sample is digested with DNase directly on the fiber matrix. Additional washing of the microspin cup removes the DNase and impurities, and the RNA is eluted from the fiber matrix with a low ionic strength buffer.
We thank Mark Dycaico for rat and mouse tissues, Michelle Cayouette for GAPDH primers and Gothami Padmabandu and Reinhold Mueller for molecular beacon RT-PCR analysis.
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