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Simple Isolation of RNA from Tissue and Cultured Cells

t 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.

Conclusions

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.).

Methods

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.

Acknowledgments

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.

REFERENCES

  1. Chirgwin
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