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