22Sample Collection and

Stability

In living cells, the mRNA content is determined by a balance of synthesis and degra-dation. When cells or tissue are removed from the body, the supply of nutrients ceasesand mRNA synthesis stops. Degradation remains and will over time reduce the mRNApool. In addition there may be induction of stress-specific genes as the blood flowceases. This has to be taken into consideration when collecting samples for DNAmicroarray analysis. The most widespread method to stop degradation is flash-freezingin liquid nitrogen and subsequent storage at −70 to −80 ◦C. This method has severaldisadvantages, however. It requires liquid nitrogen available in the operation room andit requires dry ice for shipping. Finally, samples must be homogenized before thawingto prevent resumption of mRNA degradation.

A convenient alternative is storing the sample in an ammonium sulfate solution,which precipitates the degrading RNAse enzymes. Such a solution is commerciallyavailable from Ambion (RNAlater).

The questions that remain are these: How soon must the sample be transferred to astable condition? How stable is RNA during storage? Several published studies havesought to answer these questions.

22.1 TISSUE SAMPLES

22.1.1 Stability of Tissue After Surgical Removal

Ohashi et al. (2004) conducted a time-course study to determine whether any significantmRNA degradation could be observed in human breast tissue samples after surgicalremoval and before transfer of the sample to the stabilizing solution RNAlater. RNAintegrity was judged by agarose gel electrophoresis and by RT-PCR of four genes. No

Cancer Diagnostics with DNA Microarrays, By Steen KnudsenCopyright c© 2006 John Wiley & Sons, Inc.

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150 SAMPLE COLLECTION AND STABILITY

difference in mRNA integrity was observed during the time course from 10 minutes to3 hours. The authors conclude that it is safe to transfer the tissue up to 3 hours aftersurgical removal. Fitzpatrick et al. (2002) found RNA stable in bovine reproductivetissues up to 24 hours postmortem.

Yasojima et al. (2001) found little deterioration in RNA in brain tissue up to 96hours postmortem.

Blackhall et al. (2004) studied lung cancer samples and found similar gene expres-sion profiles regardless of the time that had elapsed between resection and freezing.They studied a time interval up to 120 minutes.

22.1.2 Stability of Sample in Storage

Mutter et al. (2004) showed that a tissue sample is stable for at least 72 hours atroom temperature in RNAlater, allowing it to be shipped without cooling. They alsoshowed that samples kept at room temperature in RNAlater for 72 hours did notgive significantly different results on an Affymetrix U133A GeneChip than samplesanalyzed immediately (fresh) or samples frozen in liquid nitrogen.

Grotzer et al. (2000) found the same result for samples stored 7 days at roomtemperature in RNAlater.

As for long-term storage, Roos-van Groningen et al. (2004) found RNA stable forup to 2 months at −70 ◦C in phosphate-buffered saline (PBS), and up to 3 months at4 ◦C in RNAlater.

RNA in snap frozen tissue was found stable for 15 years at −70 ◦C (Yasojima et al.,2001).

22.1.3 Paraffin-Embedded Tissue Samples

Large tissue banks have been built with formalin-fixed paraffin-embedded tissue sam-ples (FFPE). This is not the best way of storing mRNA, but if FFPE tissue is youronly option, there are solutions. It appears that the main problem is that the transcriptsin FFPE tissue are shorter than in fresh or frozen tissue samples. This is due to chem-ical fragmentation and modification of the mRNA. Arcturus together with Affymetrixhas developed a solution to this problem. A special human array X3P is available.It is designed to focus on interrogating sequences located closer to the 3′ end of thetranscripts compared with standard GeneChip arrays. Thus the array is less sensitiveto strand breaks in the mRNA.

22.2 BLOOD SAMPLES

Tsui et al. (2002) studied the stability of blood plasma and serum. Following standardEDTA treatment, mRNA levels were stable in plasma for 24 hours at 4 ◦C. Mulleret al. (2004) demonstrated that untreated peripheral blood left at room temperatureexperiences a dramatic loss of intact RNA. A stabilizing solution added to the blood(PAXgene Blood RNA Kit) could stabilize the RNA for 3 days at room temperatureas measured by quantitative real-time PCR.

FURTHER READING 151

22.3 SAMPLE HETEROGENEITY

Blackhall et al. (2004) found considerable heterogeneity in expression levels of stressand hypoxia-activated genes in samples obtained from different areas of a lung tumorspecimen at one time point after resection. They suggest pooling from multiple sites toreduce the heterogeneity. It remains, however, to be shown whether the heterogeneityin expression levels affects the outcome of classification based on the activity of othergenes. Francis et al. (2005) addressed this issue by comparing 8–10 samples from thesame tumor to one sample obtained from 16–20 different tumors. Both for malignantfibrous histiocytoma and a leiomyosarcoma they found that samples from the sametumor were more alike than samples from different tumors in hierarchical clustering.Thus Francis et al. (2005) suggest that a single sample is representative of the tumorfor classification purposes. Schmulevich et al. (2002) came to the same conclusion ina study of leiomyosarcoma tumor heterogeneity.

Numerous single-disease studies have also measured sample heterogeneity as partof the study and found that it is not a problem for classification purposes (e.g., seeBeer et al., 2002).

22.4 SUMMARY

A stabilizing solution such as RNAlater from Ambion makes sample handling veryeasy. Shipping at room temperature in RNAlater is no problem. Note that in many ofthe above studies RNA integrity has been judged by PCR. While it does not followthat the RNA is also suitable for microarray analysis, PCR is a very good indicationof RNA integrity.

FURTHER READING

Cronin, M., Pho, M., Dutta, D., Stephans, J. C., Shak, S., Kiefer, M. C., Esteban, J. M., andBaker, J. B. (2004). Measurement of gene expression in archival paraffin-embedded tissues:development and performance of a 92-gene reverse transcriptase-polymerase chain reactionassay. Am. J. Pathol. 164(1):35–42.

Kopreski, M. S., Benko, F. A., Kwak, L. W., and Gocke, C. D. (1999). Detection of tumormessenger RNA in the serum of patients with malignant melanoma. Clin. Cancer Res.5(8):1961–1965.

Perlmutter, M. A., Best, C. J., Gillespie, J. W., Gathright, Y., Gonzalez, S., Velasco, A., Line-han, W. M., Emmert-Buck, M. R., and Chuaqui, R. F. (2004). Comparison of snap freezingversus ethanol fixation for gene expression profiling of tissue specimens. J. Mol. Diagn.6(4):371–377.