[1] RNA Extraction for Arrays

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Abstract

DNA microarrays enable insights into global gene expression by capturing a snapshot of cellular expression levels at the time of sample collection. Careful RNA handling and extraction are required to preserve this information properly, ensure sample‐to‐sample reproducibility, and limit unwanted technical variation in experimental data. This chapter discusses important considerations for “array‐friendly” sample handling and processing from biosamples such as blood, formalin‐fixed, paraffin‐embedded samples, and fresh or flash‐frozen tissues and cells. It also provides guidelines on RNA quality assessments, which can be used to validate sample preparation and maximize recovery of relevant biological information.

Introduction

DNA microarrays have enabled biologists to move from the realm of studying one gene at a time to understanding genome‐wide changes in gene expression. The value of microarray studies has been vetted through numerous studies that have linked abnormal transcript levels with many different diseases (Archacki 2004, Blalock 2004, Borovecki 2005, Dhanasekaran 2001, Glatt 2005, West 2001). Because these types of studies will be used increasingly to create and validate diagnostic and prognostic expression signatures and to support toxicological and functional studies that underlie the regulatory filings for new drug submissions, it will become increasingly important to create standardized and robust methods for sample procurement, sample processing, and data analysis. The goal of any RNA isolation procedure is to recover an RNA population that faithfully mirrors the biology of the sample at the time of collection. Problems associated with the extraction of biologically representative RNA primarily arise from the susceptibility of RNA to degradation by ubiquitous and catalytically potent RNases. For tissues and cells, protection of RNA has traditionally been accomplished by immediate lysis using high concentrations of detergents and/or chaotropic agents and organic solvents (such as TRI reagent). These methods, while effective, are complex to use at point of care and suffer from low sample throughput and poor stabilization of cellular RNA for long periods. Flash freezing of the sample in liquid nitrogen and subsequent transportation on dry ice, although effective, are impractical in most clinical settings. Finally, disease specimens can present biohazard risks to the operator and constrain sample collection, thus limiting the use of best sample handling and processing practices and compromising RNA quality.

The practicality and efficacy of RNA stabilization agents such as RNAlater to preserve the RNA in tissues, cells, and blood are gaining broad acceptance. Procedures used for collection of samples with RNAlater are simple and can be carried out in a hospital setting with minimal training. This reagent is aqueous and nontoxic and allows convenient transportation of samples at ambient temperature. However, RNAlater does not remove the biohazard risks associated with biosamples, and, as a result, all proper safety precautions should be observed. It is beyond the scope of this chapter to provide details on the risks associated and preventive measures to be taken when dealing with samples considered to be a biohazard. Several regulatory agencies offer guidelines on the safety issues and precautions that need to be addressed with such samples.

In addition to the handling of biological material, limitations can be imposed by the large amounts of RNA necessary for microarray experiments. As a result, samples such as tumor biopsies, formalin‐fixed, paraffin‐embedded (FFPE) sections, or laser microdissected samples require RNA amplification to generate adequate amounts of labeled material for microarray hybridization. The most popular and best validated approaches for amplifying RNA are based on the linear RNA amplification method developed by Eberwine (Van Gelder, 1990). This technique has been widely accepted for microarray applications and is known to preserve the original transcript ratios in the sample (Feldman 2002, Polacek 2003). In terms of RNA quality, parameters such as A260:280 measurements and Agilent RNA integrity number (RIN) are often used to gauge the quality of samples and predict their suitability for microarray studies. The minimum A260:280 or RIN number suitable for analysis varies by the array platform, number of replicates, and the experimental questions to be answered in the study.

Section snippets

Blood as a Biological Specimen

Blood is a highly desirable biosample for research and clinical studies for several reasons. First, blood is highly accessible and can be collected using relatively simple methods. Second, limited infrastructure is required to draw blood from a large number of patients. Finally, blood circulates throughout the entire body and thus is a vast reservoir of host biological information and an ideal specimen for experiments that aim to understand human physiology and disease. As a source of RNA,

Sample Collection and Capture of Leukocytes

  • 1

    Collect 9–10 ml of whole blood samples in EDTA‐containing evacuated blood collection tubes.

  • 2

    Assemble the sample tube/LeukoLOCK filter apparatus.

  • 3

    Pass blood through the LeukoLOCK filter using an evacuated tube as the vacuum source. The LeukoLOCK filter captures the total leukocyte population, while plasma, platelets, and RBCs are eliminated.

  • 4

    Flush filter with phosphate‐buffered saline (PBS) and RNAlater. Flush the filter with PBS to remove residual RBCs and then with RNAlater to stabilize leukocyte

Use of Solid Tissues for Gene Expression Analysis

In addition to blood samples, solid tissues are used routinely for gene expression analysis. Tissues collected for clinical analyses are typically biopsy specimens, which are used for histopathological testing or molecular testing using RT‐PCR. Pathological analysis of clinical tissues usually requires that the samples be fixed and embedded in paraffin to conserve the cellular architecture. As a result, specialized methods have emerged for isolating and profiling RNA from FFPE samples to more

RNA Quality Measurements for Microarray Analysis

Analysis of nucleic acid quality by absorbance spectrophotometry has been used since the inception of nucleic acid purification methodologies. Such measurements at 260 and 280 nm have been used to deduce the amount of nucleic acid and the accompanying levels of protein carryover in a sample. Absorbance ratios of 260 to 280 nm of 1.7–2.0 for RNA are often required for downstream analysis, including microarray experiments, and samples with ratios as low as 1.4 have been used successfully for gene

Conclusion

Gene expression profiling of clinical samples has become a paradigm for understanding disease etiology, pharmacogenomics and toxicological evaluations. Sample handling and the subsequent steps taken to preserve and isolate RNA can influence the quality and interpretation of microarray data dramatically. Because microarray analysis requires the use of multiple replicates to obtain statistically significant information, diligent assessments of sample procurement and preservation, RNA isolation,

Acknowledgments

The authors thank Drs. Richard Conrad, Marianna Goldrick, and Robert Setterquist who developed Ambion's RecoverAll, LeukoLOCK, and GLOBINclear products.

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