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Isolate and Analyze Total RNA from Cells Harvested by Laser Capture ,,, Microdissection



StrataPrep total RNA microprep kit for very small samples

Karen Dolter Sylvia Norman Jeff Braman

Stratagene

Isolating total RNA from laser capture microdissection samples can be challenging due to the typically small size of the samples. The StrataPrep total RNA microprep kit provides an efficient method to isolate high-quality total RNA from small cell samples. The protocol includes binding of RNA to a solid support, which eliminates the need for organic extraction and alcohol precipitation. The isolated RNA is ready for a variety of quantitative and conventional RT-PCR experiments.

Laser capture microdissection (LCM) enables researchers to collect individual cells from tissue sections.1 This method facilitates the separation of histologically different cells so that proteins, DNA, or RNA can be analyzed from the cells of choice, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. Stratagenes StrataPrep total RNA microprep kit is designed for quick and easy isolation of total RNA from 1 to 5 x 105 cells2 and is particularly useful for isolation of RNA from LCM samples. The resulting RNA can be used for both quantitative and conventional RT-PCR analysis.

RT-PCR Analysis of Varying-Abundance Messages

We isolated total RNA from LCM samples from the core of a human brain tumor section. We used this RNA to synthesize cDNA with the prostar first strand RT-PCR kit. PCR was performed using Stratagenes RT-PCR Primers for Human Gene Transcripts of Varying Abundance Levels according to the instructions.3 RNA of all abundance levels was detected in samples isolated from 200 cells. High abundance RNA was detected in a portion of the RT-PCR product equivalent to 2.5 cells, medium abundance RNA from a portion equivalent to 10 cells, and low abundance RNA from portions equivalent to 13 cells (Figure 1).

In addition, RNA isolated from malignant glioma cells using the StrataPrep microprep kit has been used in differential display experiments. These experiments identified several differentially expressed genes in the invasive rim of the tumor relative to the noninvasive tumor core.4

Conclusions

The StrataPrep total RNA microprep kit is ideal for the isolation of RNA from small cell samples harvested by LCM. The method generates pure, intact RNA with high efficiency that can be effectively amplified by conventional and quantitative RT-PCR.

Acknowledgments

The authors thank Michael Berens, Neurology Research, Barrow Neurological Institute, Phoenix, AZ for providing LCM samples.

REFERENCES

1. Emmert-Buck, M. R., et al. (1996) Science 274: 998-1001.
2. Dolter, K. and Braman J. (2000) Strategies 13: 56-58.
3. Rogers, B. and McKenzie, D. (1998) Strategies 11: 100-102.
4. Berens, M. E., et al. (2000) Proceedings of the American Association for Cancer Research 41:232, Abstract 1477.


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