Tissue and RNA Isolation

Isolation of tissues and cells

After a biological sample is isolated, its RNA becomes extremely unstable. To preserve the RNA expression pattern and to stabilize the RNA, samples should be either snap-frozen in liquid nitrogen, or, if liquid nitrogen is not immediately available, stored in RNA-stabilizing solutions, such as RNAlater™ (Qiagen).

RNA isolation

You should start your microarray experiment using RNA of the highest quality possible. Please see the section on determining RNA quality for more information.

Many RNA isolation protocols and commercial RNA isolation kits have been used successfully for isolation of high-quality RNA. Good-quality total RNA from yeast cells can be obtained using the hot phenol protocol described by Schmitt et al., 1990. Also, good results have been obtained using the TRIzol reagent (Invitrogen) for the initial isolation of RNA from a variety of mammalian tissues (Perou et al., 1999), followed by a subsequent sample clean-up, such as a phenol-chloroform extraction or using commercial products such as the RNeasy kit (Qiagen). TRIzol extraction is quick and produces a high yield of total RNA.

Total RNA versus PolyA + RNA

Good-quality microarray data have been obtained using total RNA or PolyA+ RNA as starting material for sample labeling. The results obtained from both types of sample are similar but not identical. Therefore, only samples prepared using the same sample preparation protocol should be compared.

It is advisable to first isolate total RNA and check the initial RNA quality before proceeding with PolyA+ RNA isolation. PolyA+ RNA that has been purified once using oligo-dT columns might still contain significant amounts (up to 50%) of ribosomal and other non-polyadenylated RNAs. Two rounds of purification over oligo-dT columns usually yield up to 95% pure PolyA+ RNA, but result in significant loss of material. Therefore, PolyA+ RNA isolation depends on the availability of a sufficient amount of starting material.

References:

Perou, C. M., Jeffrey, S. S., van de Rijn, M., Rees, C. A., Eisen, M. B., Ross, D. T., Pergamenschikov, A., Williams, C. F., Zhu, S. X., Lee, J. C., et al. (1999). Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A 96, 9212-9217.

Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18, 3091-3092.