Preserving RNA in Cryosections and Taking RNAlater® into Space
Your Data: RNAlater® Stories

RNAlater® is an aqueous, non-toxic tissue storage reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue samples. RNAlater eliminates the need to immediately process tissue samples or to freeze samples in liquid nitrogen for later processing. Tissue pieces can be harvested and submerged in RNAlater for storage, without jeopardizing the quality or quantity of RNA obtained after subsequent RNA isolation. RNAlater can be added to cell pellets or cells in suspension and the samples stored frozen or unfrozen.

Here we describe two unique applications of RNAlater. In the first example, Dr. Rolf Jaggi and colleagues studied gene expression in tumor and normal adjacent tissue following histological examination. They developed a technique for treating tissue cryosections with RNAlater so that the sections could be used both for pathological review and for qRT-PCR. In the second example, Dr. Anna-Lisa Paul, Dr. Robert Ferl, and colleagues demonstrated successful pairing of RNAlater with Kennedy Space Center fixation tubes, allowing safe and effective preservation of plant material on spaceflights for subsequent molecular analyses back on earth.

Preserving Intact RNA
in Cryosections with RNAlater

Rolf Jaggi*, Andrea Oberli*, Anna Baltzer*, Janine Antonov* and Hans Jorg Altermatt+

*Department of Clinical Research, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland, +Pathology Langgasse, 3012 Bern, Switzerland

E-mail: rolf.jaggi@dkf.unibe.ch

Most molecular analyses require surgical tissue samples to be snap frozen, usually in the operating room, and shipped on dry ice before further processing. Surgery departments and most pathology laboratories are generally not equipped to snap freeze and store samples at -80°C (nor to perform the molecular analyses themselves). Further, pathologists often need the entire piece of resected tumor for optimal assessment and pathological diagnoses. Consequently, tumor material for RNA isolation and molecular analysis can only be obtained after inspection by the pathologist. Dr. Jaggi and colleagues have established a protocol that treats cryosections with RNAlater resulting in high quality RNA without interfering with standard operating and pathology procedures. Molecular analysis of RNA prepared by this procedure can be closely correlated with histological analyses based on adjacent tissue.

Protocol

• Four 20 µm thick sections are transferred while maintaining low temperatures into pre-cooled 2 ml Eppendorf® tubes.

• Sections are quickly thawed and treated with 50 µl of RNAlater for 30–60 seconds.

• Excess RNAlater is removed with a micropipette.

The protocol allows wet sections to be left at room temperature for several days or stored at -20°C for longer periods. Treated sections can be shipped at ambient temperature allowing molecular diagnostics to be performed separately from routine diagnostics.

The protocol was tested on numerous breast, prostate, and lung cancer samples. Total RNA (2–9 µg) was regularly recovered depending on the size of the tumor cryosection. RNA isolated by this procedure was consistently of good quality (Figure 1) and of sufficient yield for subsequent qRT-PCR analysis. qRT-PCR was performed on nanogram or smaller quantities of RNA and the recovered RNA was sufficient for measuring the expression levels of hundreds of genes. qRT-PCR results for several genes are shown in Figure 2.

Figure 1. RNA Quality After Isolation from Treated Cryosections. Four 20 µm thick sections of prostate, breast and lung tumor material were either snap frozen (lanes labeled “snap frozen”) or treated with RNAlater® as described in the protocol (lanes labeled “RNAlater”). Alternatively, sections were homogenized in lysis buffer (lanes labeled “LB”) or transferred to an RNase free microscopic slide, fixed with cold 70% acetone for 2 x 5 min and air dried (lanes labeled “acetone”). After three days at room temperature, sections were homogenized and RNA was isolated (RNeasy®; Qiagen®). RNA was separated on an Agilent® 2100 bioanalyzer. Shown are gel-like pictures of the resulting RNA. The band between 28S and 18S rRNA most likely represents contaminating DNA. (Courtesy of Dr. R Jaggi, University of Bern. Tumor samples were obtained from Tumorbank Bern.)

Figure 2. qRT-PCR of RNA Isolation from Treated Cryosections. Total RNA (150 ng) was reverse transcribed using random primers, and the resulting cDNA (1.6 ng) was used for quantitative PCR with TaqMan® MGB probes specific for GAPDH, IGFBP5, and ERBB2. Similarly, cDNA (8 pg) was used for detection of 18S rRNA. Shown are quantification plots for 18S rRNA, GAPDH, and IGBP5 (A) and threshold values (Ct) for 2 independent breast cancer samples (B). Samples were snap frozen, or treated with RNAlater®, LB buffer, or 70% acetone as described in the legend for Figure 1. Fixed samples were stored at room temperature for three days before RNA isolation. NTC = no template control (PCR in the absence of cDNA). (Courtesy of Dr. R Jaggi, University of Bern).

RNA Storage that is Out of this World!

Anna-Lisa Paul*, Howard G Levine*#, William McLamb*+, Kelly L. Norwood*+, David Reed*+, Gary W Stutte*#, H William Wells*+, Robert J Ferl*

*Department of Horticultural Sciences, University of Florida, Gainesville, FL32611, USA, #Dynamac Corporation, Kennedy Space Center, FL 32899,USA, +Bionetics Corporation, Kennedy Space Center, FL 32899,USA.

Email: robferl@ufl.edu

Plant Molecular Biology in the space station era: Utilization of KSC fixation tubes with RNAlater® (2005) Acta Astronautica (56):623-628.

Scientists at the University of Florida are working with NASA studying plant biology, as plants are central components of the advanced life-support system that will support long-term occupation of orbital facilities and extended spaceflight missions. Plants are also an excellent model system to study the effects of abiotic stress. Most plant research requires that some of the sample collection be conducted while in orbit, which provides material that is uncompromised by landing and post-flight handling delays.

In flight, sample harvest and proper storage of harvested samples are not trivial. Of significant concern, is the harvesting and stowing of samples obtained during spaceflight experiments to maximize high quality RNA. Snap freezing in liquid nitrogen and storage at –80°C is very effective in maintaining protein and nucleic acid integrity. However, current orbital cryogenic freezer designs hold temperature for a maximum of 35 days, making them suitable only for short spaceflights.

Storage of samples in chemical fixatives like RNAlater provides several options without compromising RNA integrity. Figure 3 shows RNA isolated from Arabidopsis and wheat samples exposed to a variety of storage conditions. The data simulate those obtained from harvesting plants with RNAlater during both short and long duration missions. The storage condition of –20°C mimicked the –25°C compartment of the freezers used on board. The presence of equimolar amounts of cytoplasmic and plastid rRNA indicate that there was no degradation of the RNA sample.

Figure 3. Plant Samples Stored in RNAlater® for Long and Short Spaceflight Times. RNA samples are shown from Arabidopsis(A) and Wheat (B) plants. The individual lanes show RNA from differentially treated samples.  Panel A shows RNA from Arabidopsis samples that were treated as follows: 1: placed directly into liquid nitrogen, held at –80oC for 10 days 2: slow freeze at –80°C, held at –80°C for 10 days, 3: slow freeze at –20°C, held at –20°C for 10 days, 4: 4°C plus RNAlater, held at 4°C for 10 days, 5: room temperature plus RNAlater, held at room temperature for 10 days, 6: –20°C plus RNAlater, held at –20°C for 10 days, 7: Held at 4°C for 10 days. Panel B shows RNA from wheat samples that were treated as follows: 1: isolated fresh, 2: slow freeze at –20°C, held at –20°C for 10 days, 3: 4°C plus RNAlater, held at 4°C for 60 days, 4: –20°C plus RNAlater, held at –20°C for 60 days, 5: Sample from ISS mission profile (PESTO-Dr. Gary Stutte) stored in RNAlater at various temperatures, (Courtesy of Dr. A-L Paul and RL Ferl, University of Florida).

Scientific Contributors

Jon Kemppainen, Gary Latham • Ambion, Inc.

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