Article

Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays

Brigham and Women's Hospital, Boston, Massachusetts, United States
BMC Genomics (Impact Factor: 3.99). 11/2004; 5(1):88. DOI: 10.1186/1471-2164-5-88
Source: PubMed

ABSTRACT

Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted.
14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance.
Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment.

Download full-text

Full-text

Available from: David B Finkelstein
  • Source
    • "Over long distances, the sample may thaw, and multiple freeze-thaw cycles and prolonged exposure to increased temperatures must be avoided, as these conditions promote degradation of labile RNA samples [3]. There are new technologies that help preserve RNA at room temperature, such as RNALater® (Ambion, Carlsbad, CA, USA), which helps preserve sample tissues for further RNA extraction, and RNA stable® (Biometrica, San Diego, CA, USA), which keeps isolated RNA in anhydrobiosis at room temperature for weeks [3] [4]. However, these methods are costly and need to be in hand at the laboratory at the moment of use. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Gaseous fumigants are commonly employed to control fungal decay of cold-stored grapes. So far it is not clear if these fumigants, beside the direct interaction against fungal structures, induce transcriptional responses of defensive markers. In order to contribute to understand the mechanisms by which these fumigants exert their effect, it was studied the influence of ozone (O3 ) and sulfur dioxide (SO2 ) on the decay caused by Botrytis cinerea, and the quality and expression of the defense related genes chitinase, β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) in the table grape cultivars 'Redglobe' and 'Sugraone'. The application of SO2 or O3 delayed both table grape cultivars decay caused by B. cinerea compared with the inoculated control. O3 treatments altered weight loss, firmness and shatter in both cultivars. Significant up-regulation of chitinase and β-1,3-glucanase were observed in SO2 -treated 'Redglobe' berries stored at 2 °C. O3 treatment transiently increased the expression of chitinase and PAL in 'Redglobe' and 'Sugraone' berries, respectively. Ozone and sulfur dioxide treatments can influence the expression patterns of PAL, chitinase and β-1,3-glucanase to different extents in different grape cultivars and under different exposure conditions. The upregulation of these genes may be involved in the mechanism by which these fumigants inhibit the decay caused by pathogenic fungi. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jun 2015 · Journal of the Science of Food and Agriculture
  • Source
    • "Over long distances, the sample may thaw, and multiple freeze-thaw cycles and prolonged exposure to increased temperatures must be avoided, as these conditions promote degradation of labile RNA samples [3]. There are new technologies that help preserve RNA at room temperature, such as RNALater® (Ambion, Carlsbad, CA, USA), which helps preserve sample tissues for further RNA extraction, and RNA stable® (Biometrica, San Diego, CA, USA), which keeps isolated RNA in anhydrobiosis at room temperature for weeks [3] [4]. However, these methods are costly and need to be in hand at the laboratory at the moment of use. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant tissues must be preserved in their collection state, especially for genome-wide expression profile studies. Lyophilization is a feasible, affordable tool when fresh tissues cannot be shipped at ultralow temperatures from their origin to the place of analysis. In this study, the total RNA quality of dormant grapevine buds (Vitis vinifera L. cv. ‘Flame Seedless’) of freeze-dried samples stored at room temperature conditions was evaluated and compared to that of cryopreserved (-80°C) grapevine buds.
    Full-text · Article · Feb 2015 · Electronic Journal of Biotechnology
  • Source
    • "The most common technique used for preserving the integrity of the samples has been freezing7. However, other methods for stabilizing the samples have been used14, and at present there are products on the market that promise to stabilize the DNA immediately after the immersion of the biological sample into the product6,10,11,13. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Objective: The present study evaluated the use of a reagent to stabilize the DNA extracted from human dental tissues stored under different temperature conditions and time intervals. Material and methods: A total of 161 teeth were divided into two distinct groups: intact teeth and isolated dental pulp tissue. The samples were stored with or without the product at different time intervals and temperature. After storage, DNA extraction and genomic DNA quantification were performed using real-time PCR; the fragments of the 32 samples that represented each possible condition were analyzed to find the four pre-selected markers in STR analysis. Results: The results of the quantification showed values ranging from 0.01 to 10,246.88 ng/μL of DNA. The statistical difference in the quantity of DNA was observed when the factors related to the time and temperature of storage were analyzed. In relation to the use of the specific reagent, its use was relevant in the group of intact teeth when they were at room temperature for 30 and 180 days. The analysis of the fragments in the 32 selected samples was possible irrespective of the amount of DNA, confirming that the STR analysis using an automated method yields good results. Conclusions: The use of a specific reagent showed a significant difference in stabilizing DNA in samples of intact human teeth stored at room temperature for 30 and 180 days, while the results showed no justification for using the product under the other conditions tested.
    Full-text · Article · Jul 2014 · Journal of applied oral science: revista FOB
Show more