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Assessing a novel room-temperature RNA storage medium for compatibility in microarray gene expression analysis
 
Gilberto E. Hernandez, Tony S. Mondala, and Steven R. Head
DNA Array Core Facility, The Scripps Research Institute, La Jolla, California, USA
BioTechniques, Vol. 47, No. 2, August 2009, pp. 667–670
Full Text (PDF)
Abstract

RNA integrity is a critical factor in obtaining meaningful gene expression data. Current methodologies rely on maintaining samples in cold environments during collection, transport, processing, and storage procedures, which are also extremely time-sensitive. Several RNA storage products are commercially available to help prevent degradation during the handling and storage steps; however, samples must be kept cold for optimal protection. We have evaluated a novel RNA storage medium based on anhydrobiosis for stabilizing and protecting samples from degradation at room temperature that are intended for use in microarray analysis. Samples were stored dry at room temperature for various time periods to assess any degradation or loss of activity as compared with frozen control samples. Recovered samples were used directly for analysis without further purification and exhibited no interference or inhibition in downstream applications. Comparison of gene expression profiles indicate no significant differences between freezer-stored control samples and those kept at room temperature protected in the RNA storage medium. The quality of recovered RNA was confirmed using spectrophotometry and Bioanalyzer analysis and was identical to control samples. The ability to stabilize RNA samples at ambient temperatures for extended time periods will have tremendous use, particularly for sample shipment to core facilities.

Introduction

The use of microarray technology to measure changes in gene expression profiles has become a standard tool for studying biological models of disease, growth, and development, and is increasingly being used in the field of clinical research. While technology advances continue to increase the sensitivity of expression profiling assays such as quantitative real-time PCR and microarray analysis, the fundamental baseline for generating quality data rests on the quality and integrity of the RNA sample.

Special care is needed when working with RNA due to its chemical instability and the ubiquitous presence of RNases in the laboratory. Commercially available products, such as RNAlater (Ambion, Austin, TX, USA), have enabled successful preservation of tissues and cells for long-term storage at cold or freezing temperatures prior to RNA purification 1,2,3,. Room-temperature storage (18–25°C) in RNAlater is possible for up to 7 days according to manufacturer's instructions, although significant mRNA degradation was detected after 3 days of room-temperature storage of rat liver samples soaked in RNAlater 2,. Purified RNA samples are handled minimally and kept cold at all times unless otherwise directed. Samples are typically stored frozen (−20°C, −80°C, or under liquid nitrogen) in RNasefree solutions (e.g., DEPC-treated water, TE buffer or other commercially available RNA storage solutions) and thawed on ice only when needed. Multiple freeze-thaw cycles and prolonged exposure to increased temperatures are avoided as these conditions serve to promote degradation of precious and labile RNA samples. Despite all the precautions taken to maintain what is typically referred to as cold chain logistics, the integrity of a sample is often compromised using current cold storage methodologies, particularly during long-term storage or sample transport, where shipment delays and suboptimal packaging are a common occurrence. There is a critical need for technologies providing simple room-temperature storage of biological samples that do not require cold chain logistics or complicated sample recovery protocols.

A novel storage medium designed specifically to preserve and protect purified total RNA samples stored dry at room temperatures is commercially available. RNAstable (Biomatrica, San Diego, CA, USA) is a synthetic matrix that was developed based on the principle of anhydrobiosis, a biological mechanism employed by some multicellular organisms that enables their survival while dry for >100 years 4,. The matrix forms a thermostable barrier around RNA during the drying process to protect samples from degradation during storage at ambient temperatures. Samples are ready for immediate analysis following a single rehydration step, eliminating the need for further downstream purification. We have evaluated the use of RNAstable for storage of purified total RNA samples derived from various human tissues for compatibility in microarray analysis for gene expression profiling. The quality of the RNA was assessed by RNA Integrity Number (RIN) score [on an Agilent 2100 Bioanalyzer (Palo Alto, CA, USA)], yield of total RNA, OD260/280 ratio, and by the microarray QC parameters of percent present and 3′/5′ ratios of GAPDH and β-actin probe sets on an Affymetrixgene expression array (GLYCOv3 array; Consortium for Functional Glycomics). Results indicate that samples stored for ≤5 weeks at room temperature while protected in RNAstable in a desiccating environment are indistinguishable from control samples stored frozen at −80°C. Samples rehydrated from storage in RNAstable were used directly without further purification and did not exhibit any interference or inhibition during microarray analysis. Use of RNAstable for room-temperature storage of purified RNA can reduce reliance on current cold storage methodologies. The storage medium is particularly suited for sample transport, as unexpected delays will not damage the RNA. Samples can be prepared with minimal effort and shipped without expedited delivery fees or bulky dry-ice containers. Maintaining a controlled humidity (i.e., desiccating) environment for optimal stabilization and protection of RNA is easily achieved by storing dried samples in packaging that includes a desiccant pack supplied by the manufacturer. Such applications will have particular use for sample transport to core array facilities.

Materials and Methods

RNA preparation

Purified human liver and kidney total RNA (100 µg) were purchased from Ambion and diluted to 250 ng/µL. The quality of the RNA prior to any treatment was assessed using an Agilent 2100 Bioanalyzer and the RIN was determined to be 9.6 and 7.9 for the human liver and kidney total RNA, respectively. Aliquots (20 µL) were applied directly into 1.5-mL tubes containing RNAstable (supplied with the kit) and dried in a Savant Integrated SpeedVac System ISS110 vacuum concentrator (Instrument Inc, Holbrook, NY, USA) without heat for 30 min following manufacturer's instructions prior to storage at room temperature for various time periods. Dried samples were then stored at room temperature on the benchtop inside a sealed-moisture barrier bag including a desiccant pack (both are included with the kit), following manufacturer's guidelines. Aliquots (20 µL) used for frozen control samples were placed into 1.5-mL Eppendorf tubes (Cat. no. 1615-5510, USA Scientific Inc, Orlando, FL, USA) and stored at −80°C until ready for use. Samples were recovered from storage in RNAstable by rehydrating with 20 µL RNase-free water and used directly for analysis without further purification. RNA quantification was determined by spectrophotometry using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA). The OD260/280 ratio was used to evaluate the purity of the nucleic acid samples and the quality of the extracted total RNA was determined with the Agilent Bioanalyzer.

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