High-quality RNA extracted from biopsied samples dehydrated and stored dried at room temperature without chemical preservation for up to 3 months as evidenced by RT-PCR results.

TECHNICAL ARTICLE

High-quality RNA Extracted From Biopsied Samples Dehydrated and Stored Dried at Room Temperature Without Chemical Preservation for up to 3 Months as Evidenced by RT-PCR Results Theodore R. Sadler, PhD* and Ani C. Khodavirdi, PhDw

Abstract: Handling and maintenance of biological tissues for nucleic acid and/or protein analysis has long been a challenge because of the perceived instability of these molecules at room temperature if not preserved or processed. Structural damage and compromised integrity of aforementioned biomolecules subsequent to preservation have also posed difficulties in their use in research. The development of technologies employing nonfixative methods with the capability to store at room temperature have been of growing interest. Our previous publication exploring preservation of proteins by desiccation challenged the convention of their unstable nature. Herein, we report the results of quantitative and qualitative analyses of RNA from tissue samples that were desiccated and stored at room temperature for up to 3 months. Our results indicate that viable RNA can be obtained from dehydrated ex vivo tissue samples that have been stored at room temperature. Key Words: RNA, RT-PCR, anhydrobiosis, desiccant, room temperature storage (Appl Immunohistochem Mol Morphol 2015;23:456–461)

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ne of the critical biomolecules used for diagnostic and research purposes is RNA. Per conventional beliefs, the RNA structure is highly susceptible to degradation if not cryogenically maintained or treated with chemicals for keeping at normal environmental conditions. According to the literature, examinations of unpreserved samples identified nucleases as the main culprit for RNA degradation as confirmed by loss of signal.1–4 Like many other enzymes, nucleases primarily depend on the presence of water to cleave bonds, subsequently degrading biological molecules. To disable such biochemical reactions and preserve structural and functional integrity under normal environmental conditions, RNA is treated Received for publication October 15, 2013; accepted April 6, 2014. From the *Department of Science, Concordia University, St. Paul, MN; and wDepartment of Pathology, USC Keck School of Medicine, Los Angeles, CA. The authors declare no conflict of interest. Reprints: Theodore R. Sadler, PhD, 4336 York Avenue South, Minneapolis, MN 55410 (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.

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with stabilizing agents or chemical fixatives such as formaldehyde,5 which ironically renders it damaged because of cross-links and other structural alterations.5–8 Thus, it has been difficult or nearly impossible to generate meaningful gene expression profiles from RNA preserved with such chemicals.6,7,9,10 The negative impact of commonly used preservatives and fixative agents on essential biomolecules such as nucleic acids and proteins has been the primary driver for development of technologies employing alternative storage of nonfixed biological specimens at room temperature.2,3,5,7,9–12 Other potential benefits of such technologies include reduction in exposure to toxic and/or carcinogenic chemicals, elimination of expensive refrigeration systems, reduction in cost and waste generation/disposal, and reduction in overall environmental impact.6,9,10 Consequently, the concept of anhydrobiosis, which is an ametabolic state exemplified by desiccation, has become an area of great interest and focus because of its central role in nonfixative, room temperature storage.6–8,13 During cellular desiccation (elimination of water) as observed in simple unicellular organisms, specialized carbohydrates are synthesized to first stabilize intracellular biomolecules, and next, to vitrify the cell for protection from potential damage.13 Mimicking the function of these unique carbohydrates, various aqueous-based compounds have been developed for commercial use.7,8 Their application requires a 3-step process, wherein the sample is coated and then completely dehydrated by a vacuum centrifuge or similar device.7,8 When ready for use, the coating is removed by immersing the sample in water. Although it is believed that this technique could preserve samples for up to 120 years and should be compatible with modern molecular methods, for instance reverse transcription-polymerase chain reaction (RT-PCR),6,8,13 it has certain limitations: The dehydration step could last 2 to 3 days, during which time the cells are susceptible to damage; and The step-wise process could be cumbersome, especially for large number of samples. We previously reported sustained stability of proteins extracted from brain tissue sections that were desiccated and stored at room temperature under ultra low (under 10% relative humidity), dry air conditions over a

Appl Immunohistochem Mol Morphol

Volume 23, Number 6, July 2015

Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.



period of 6 months.12 The 2 comparators were nondesiccated tissue sections stored at room temperature and fresh-frozen tissues. Samples were not treated with chemical fixatives, coatings, or protease inhibitors. This work illustrated that proteins, also thought to be susceptible to degradation at room temperature without chemical fixation, were not as sensitive. Evaluation of our desiccated, room temperature-stored protein samples exhibited minimal denaturation and sustained epitope specificity as demonstrated by gel stains and by immunoblots (Western blot) and immunohistochemical analysis (fluorescent-based and peroxidase-based) stains, respectively.12 To complement our original study exploring protein stability, we sought to characterize the stability of RNA isolated from nonfixed, uncoated, desiccated tissue samples stored at room temperature. Micke et al10 asked a similar question about the “unstable” nature of RNA; they examined fresh, unfrozen tissue samples left at room temperature for 16 hours and found little degradation.10 We present the findings of our study with tissue samples stored at room temperature for up to 3 months.

MATERIALS AND METHODS Equipment and Tool Preparation All equipment and surgical tools that came in direct contact with tissue samples, fresh and postdry storage, were either confirmed to be nuclease-free by the manufacturer or were made nuclease-free by a series of sterilization steps. These steps included thoroughly washing and rinsing all equipment, followed by soaking in diethylpyrocarbonatetreated ultra pure water (Fisher Scientific, Pittsburgh, PA), autoclaving (as necessary), and finally placing in a drying oven for a minimum of 12 hours. This method provided the best means of minimizing DNase and RNase activity.1–3,7

Tissue Acquisition, Designation, and Storage Gallus gallus liver was selected as the tissue of choice because of its availability and high concentration of both DNA and RNA. Nonvivum adult male G. gallus (n = 4) (RodentPro.com LLC; Inglefield, IN) were received from the vendor in an insulated Styrofoam container filled with dry ice. These animals were euthanized by the vendor, frozen immediately, and shipped within 24 hours. Upon receipt, the animals were allowed to thaw for 8 to 12 hours in a 41C refrigerator, following which whole livers were removed. Using a scalpel and forceps, 6 biopsies of approximately 5 5 5 mm in size were obtained from randomly selected areas of each whole liver (n = 4), for a total of 24 samples. These samples were further randomly sorted into 1 of 6 timepoints for analysis: 8 hours, 1 day, 1 week, 2 weeks, 1 month, and 3 months. Samples were allocated in triplicate with a fourth set as back-up but because of resource constraints, only one sample from each timepoint was selected at random for RNA analysis. Each sample was placed onto weighing paper (Fisher Scientific) and its mass was recorded (Mettler-Toledo m/n MS403S; Mettler-Toledo, Columbus, OH). Next, each sample with its weighing paper was placed in the center of Copyright

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High Quality RNA Extracted From Biopsied Samples

a 60 15 mm plastic Petri dish (Fisher Scientific) that contained CaCl2(s) desiccant evenly spread along the inner wall (Fisher Scientific). The mass of desiccant used was approximately 5 times the mass of the sample, as determined by trial and error to dry-out the sample as efficiently as possible. Each Petri dish was sealed with Parafilm (Pechiney Plastic Packaging, Chicago, IL) and stored at room temperature (201C) for prespecified timepoints. This setup mimicked an ultra low, dry air environment within the Petri dish. Three samples intended as controls were obtained as biopsies of approximately 5 5 5 mm in size upon thawing of the livers. They were immediately processed for RNA extraction, and purified aliquots were stored under cryogenic conditions. One sample was randomly selected for analysis.

RNA Extraction and Quantification At prespecified timepoints, samples were carefully removed from their individual sealed storage containers (Petri dish) and weighed. Table 1 summarizes the initial mass, final mass, and % change in mass of all 6 tissue samples. Immediately following weight measurement, each sample was ground into a fine powder using a sterilized mortar and pestle. The ground sample was rapidly transferred to a 1.5 mL nuclease-free microcentrifuge tube (Fisher Scientific). Using a commercial extraction kit (Total RNA Purification System; Invitrogen, Grand Island, NY), RNA was isolated from the pulverized sample. This step required rehydration of the dried sample, which was achieved by the addition of the activated extraction buffer from the extraction kit and aspiration of the sample with a 27 G needle up to 10 times until fully dissolved. Once the RNA was isolated and collected in nuclease-free water, the total quantity and overall quality was determined by UV Spectrophotometer (Beckman DU-64 UV Spectrophotometer with Nucleic Acid SoftPac, Rev A; Beckman-Coulter, Brea, CA). Each sample was divided into 3 aliquots (1 ng each) and either immediately used or stored at 801C.

cDNA Generation and RT-PCR To verify the integrity of RNA extracted from desiccated tissue sections, each purified RNA sample was reverse transcribed using a commercial kit (Invitrogen SuperScript III One-Step RT-PCR with Platinum Taq; Invitrogen) and amplified (Bio-Rad MJ Mini Thermal

TABLE 1. Tissue Mass Measurements Duration of Desiccation 8h 1d 1 wk 2 wk 1 mo 3 mo Mean

Initial Mass (g)

Final Mass (g)

Change (%)

0.092 0.173 0.131 0.162 0.153 0.167

0.027 0.028 0.033 0.042 0.043 0.049

70.7 83.8 74.8 74.1 71.9 70.7 74.3

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DISCUSSION

Cycler; Bio-Rad, Hercules, CA) with primers (Eurofins MWG Operon, Huntsville, AL) from previously published data.14,15 One pair of primers amplified GAPDH, whereas the other amplified b-actin. The primer sequences and corresponding PCR conditions are summarized in Table 2. After RT-PCR, 2 mL of the amplified product with 6 dye (Promega, Madison, WI) was loaded onto a 2% TAE ethidium bromide-doped agarose gel. DNA ladders of 1 kb and 10 kb (Promega) were used to confirm GAPDH and b-actin amplicon size, respectively. The gel ran at 100 V for 60 minutes (Bio-Rad Power Pac 3000; Bio-Rad) and visualized with Bio-Rad Universal Hood II, IBM desktop computer with QuantityOne Software version 4.6.7 (Bio-Rad).

Our goal was to desiccate tissue samples for prespecified periods of time and evaluate the RNA quantity and quality. As we could neither measure the amount of water in the samples at baseline nor the amount of water lost because of desiccation, we assessed tissue mass at baseline and at prespecified timepoints to infer the extent of desiccation. Acquiring accurate mass data from dried samples proved somewhat challenging. The samples strongly adhered to the weigh paper and, during removal, small fragments of the tissue were left behind. In some instances, not all tissue was retrieved, resulting in higher values for percent change. The texture of the tissues was not of primary interest; however, we noted wax-like consistency for “younger” samples, whereas tissues stored for 1 week or longer were brittle and could be easily crushed into a fine powder with a mortar and pestle. The relevance of this observation is currently inconclusive because of lack of comparator tissue. Analysis of the desiccated samples (except the 3-month sample) revealed a surprising trend: even after 1 month, RNA purity remained as high as the fresh tissue (260 nmAbs:280 nmAbs = 2.2) with little protein or particulate contamination (data not shown). Although total RNA and mRNA quantities decreased over time, the amount of mRNA in each sample was sufficient for cDNA generation. The extracted RNA was not analyzed for 28S:18S rRNA banding because of variability of these ribonucleoprotein; they rarely demonstrate a true ratio of 2.0 and, moreover, they exhibit different degrees of stability that may not reflect the true RNA integrity.4,11 Instead, sustained RNA integrity and quality was confirmed by amplification of 2 housekeeping genes, GAPDH and b-actin. With the former, signal intensity of desiccated tissues was comparable with the fresh one, keeping in mind that the amounts of loaded samples were controlled for volume, not quantity. To replicate these results as well as to confirm the specificity of the amplified genomic material (cDNA vs. genomic DNA), RT-PCR for b-actin was performed using primers known to only amplify cDNA. Similarly, the loaded amounts were controlled for volume, not quantity. Thus, variation in

RESULTS After exposure to desiccant, the tissue masses decreased by an average of 74.3% as compared with initial wet mass (Table 1). The amount of total RNA extracted from tissues desiccated for 8 hours, 1 day, and 1 week was comparable with total RNA quantity from the fresh sample (Fig. 1A). However, a decrease of approximately 25% in total RNA was observed in samples after 2 weeks. The changes in mRNA quantities followed a trend similar to that of total RNA. Spectrophotometric analysis of RNA purity, as determined by the ratio of 260:280 absorbance, demonstrated no difference between RNA extracted from fresh tissues versus desiccated tissues stored at room temperature for 1 month. Specifically, samples desiccated for 8 hours, 1 day, 1 week, 2 weeks, and 1 month had a 260 nmAbs:280 nmAbs value of 2.2, same as the fresh sample (Fig. 1B). There was a slight decrease in 260 nmAbs:280 nmAbs value (2.1) for the 3-month desiccated sample. Amplification of GAPDH and b-actin by RT-PCR produced signals corresponding to the respective amplicons (Figs. 2A, B). Evidenced by nonspecific bands in the GAPDH RT-PCR, small amounts of genomic DNA contamination was present in the fresh, 1-week desiccated, and 3-month desiccated tissue samples. Similarly, amplification of b-actin had contamination in the fresh and 3-month desiccated samples.

TABLE 2. GAPDH and b-actin Primer Sequences and Corresponding RT-PCR Conditions b-Actin Forward 50 -TATCCGTAAGGATCTGTATG-30 Reverse 50 -ATCTCGTCTTGTTTTATGCG-30 Steps 1 2 3a 3b 3c 4

GAPDH Forward 50 -ACGCCATCACTATCTTCCAG-30 Reverse 50 -CAGCCTTCACTACCCTCTTG-30

Temperature (1C)

Time

Cycle(s)

Temperature (1C)

Time

Cycle(s)

50 98 98 51 72 —

30 min 1 min 10 sec 15 sec 45 sec —

1 1

50 94 94 52 72 68

30 min 2 min 30 sec 30 sec 45 sec 7 min

1 1

30 —

25 1

RT-PCR indicates reverse transcription-polymerase chain reaction.


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FIGURE 1. UV spectrophotometer assessment of RNA quantity and quality. A, Measurement of total RNA and estimated mRNA. The estimated mRNA quantity was calculated from known values of mRNA lying between 5% and 10%, that is, 7.5%, in eukaryotic animal cells. Duration of storage with desiccant (prespecified timepoints) is shown on the x-axis, and quantity of RNA in nanograms is displayed on the y-axis. B, Determination of RNA quality. Duration of storage with desiccant (prespecified timepoints) is shown on the x-axis, and the ratio of UV wavelength absorbance at 260 and 280 nm are displayed on the y-axis. UV indicates ultraviolet.

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FIGURE 2. RT-PCR results for GAPDH and b-actin. A, Visualization of GAPDH amplicon (578 bp) on 2% agarose gel. Lane 1, 1 kbp DNA ladder; lane 2, blank; lane 3, RNA extract from fresh tissue; lane 4, RNA extracted from tissue desiccated for 8 hours; lane 5, RNA extracted from tissue desiccated for 1 day; lane 6, RNA extracted from tissue desiccated for 1 week; lane 7, RNA extracted from tissue desiccated for 2 weeks; lane 8, RNA extracted from tissue desiccated for 1 month; and lane 9, RNA extracted from tissue desiccated for 3 months. B, Visualization of b-actin amplicon (338 bp) on 2% agarose gel. Lane 1 = 10 kbp DNA ladder; lane 2, blank; lane 3, RNA extract from fresh tissue; lane 4, 8 hours RNA extract; lane 5, RNA extracted from tissue desiccated for 1 day; lane 6, RNA extracted from tissue desiccated for 1 week; lane 7, RNA extracted from tissue desiccated for 2 weeks; lane 8, RNA extracted from tissue desiccated for 1 month; and lane 9, RNA extracted from tissue desiccated for 3 months. RT-PCR indicates reverse transcription-polymerase chain reaction.

signal intensity could be attributed to different concentrations of loaded samples. We recognize that this study was not without limitations. Our analysis was based on a small sample size from a single tissue type. Although the population for each timepoint was low in number (n = 1), results throughout the 3-month period has warranted a more extensive investigation with larger sample size and various tissue types. In addition, RNA stability and its handling and storage methodologies must be evaluated at longer timepoints. Future analyses would also include RIN or RNA integrity number measurements for assessing RNA integrity and real-time PCR (qPCR) for quantifying amplified sequences of interest. Despite the aforementioned limitations of which we were aware in advance of study initiation, we have demonstrated using basic research methods that intact RNA can be extracted in sufficient amounts from ex vivo tissue samples, desiccated and stored at room temperature for up to 3 months. Moreover, our results have shown that RNA extracts from these desiccated tissues can produce viable cDNA. Challenging conventional beliefs of RNA stability, these findings have established the short-term stability of RNA from desiccated biological samples stored at room temperature and have warranted further investigation of long-term RNA stability.


ACKNOWLEDGMENT The authors thank Christine Mennicke for her technical assistance gathering data for this study.

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