An Optimized Procedure for Extracting RNA from Human Ocular Tissues
Wang, W-H., McNatt, L. G., Shepard, A. R., Jacobson, N., Nishimura, D. Y., Stone, E. M., Sheffield, V. C., and Clark, A. F. (2001) Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative PCR. Mol Vis 7: 89-94. (Article)

Researchers often need to determine gene expression patterns in tissue samples as part of their studies. Gene expression analysis typically requires RNA isolation. RNA degradation, however, has been a major concern for tissues that are isolated away from the laboratory. Traditional methods of "in-tissue" RNA preservation — cooling of tissues on ice or sample freezing — have been met with varying degrees of success. The RNA preserving solution, RNAlater, on the other hand, has been shown to perform better than traditional methods. This product, along with Ambion's RNA isolation kits, has proven to be an invaluable tool for many scientists.

A recent paper by Wang and coworkers illustrates this assertion. These researchers were interested in determining the ocular-spatial expression of FOXC1, a gene associated with the eye disease glaucoma. Initially, the group tried to determine the best method of in-tissue RNA preservation. Human eye tissues were either stored on ice, on dry ice, or kept in RNAlater. The quality of the isolated RNA was subsequently compared between the samples. Use of RNAlater consistently gave rise to higher yields and a better quality of isolated RNA.

Next, the researchers shifted their attention to finding the best way of isolating pigment-free RNA. Pigments were known to interfere with RT-PCR, the method by which the researchers were analyzing expression levels of the FOXC1 gene. Two RNA isolation kits, representing two different isolation methods, were deployed: the ToTALLY RNAª Kit (based on the guanidinium isothiocyanate method) and the RNAqueousª-4PCR Kit (a silica-based filter-binding method). Both methods yielded intact RNA, but the RNAqueous-4PCR Kit was also effective at removing the pigment.

With an optimized procedure for extracting RNA from human ocular tissues, Wang and colleagues were able to analyze FOXC1 gene expression in several ocular tissues using real-time PCR. They found that FOXC1 expression levels were highest in the eye's trabecular meshwork, followed by the optic nerve head, ciliary body, cornea, iris, retina, and the lens.

RNAlater and Histology
Florell SR, Coffin CM, Holden JA, Zimmermann JW, Gerwels JW, Summers BK, Jones DA, Leachman SA (2001) "Preservation of RNA for Functional Genomics Studies: A Multidisciplinary Tumor Bank Protocol." Mod Pathol 14:116-128.

Many of our customers have inquired about the suitability of using RNAlater as a "holding solution" to preserve both the RNA and the histology of tissue samples collected from clinical sources. This would enable the recovery of intact RNA from samples held in reserve for pathologists.

Drs. Scott Florell and Sancy Leachman at the Huntsman Cancer Institute recently completed a blinded study in which 2 pathologists compared human tissue sections that were either immediately processed for histology (fixed in formalin and embedded in paraffin, or frozen sectioned) to samples stored in RNAlater, rinsed, and then processed for histology. Their results indicate that morphological detail and staining characteristics were identical for the 2 groups of samples. An example of their findings is presented in Figure 1. In addition to excellent staining and preservation of morphological detail, many immunohistological stains performed equally well in the RNAlater preserved samples, suggesting preservation in RNAlater caused no damage to cellular epitopes.

Figure 1. Histology of RNAlater Preserved Tissue. RNAlater preserved samples are suitable for histology and provide excellent morphological detail. Sections made from RNAlater preserved material were indistinguishable from slides made from untreated samples when examined for standard histological criteria. Panel A. H&E stained, frozen section of human skin preserved for one week in RNAlater prior to processing. Panel B. Stained as above but formalin-fixed, paraffin-embedded section of human skin preserved for one week in RNAlater prior to processing.

RNAlater Facilitates mRNA Expression Analysis
Grotzer, M. A., Patti, R., Geoerger, B., Eggert, A., Chou, T. T., and Phillips, P. C. (2000) Biological stability of RNA isolated from RNAlater-treated brain tumor and neuroblastoma xenografts. Med Pediatr Oncol 34: 438-442.

Recently, it was reported that RNAlater-fixed tissues were compatible with mRNA expression analysis of tumor samples. mRNA expression is used as a prognostic factor for the definition of risk groups in clinical trials involving neuroblastoma tumors. In the search to find biological prognostic factors for other cancers, RNA expression analysis must be completed on numerous tumors. Until now multi-institutional studies of tumor samples have been difficult because the rigid conditions under which tissue must be stored in order to prevent RNA degradation hamper collection and mailing. Traditionally, tumor biopsies are snap-frozen in liquid nitrogen and stored at -70¡C prior to RNA isolation. Samples fixed in RNAlater do not need to be kept frozen but can be stored at room temperature for up to a week or at 4°C for a month. Thus, in principle, samples treated with RNAlater would be easier to ship and would still maintain RNA integrity. This study evaluated Ambion's RNAlater Tissue Collection: RNA Stabilization Solution as a storage solution for tumor samples from which RNA was to be isolated.

CNS tumor tissue and human neuroblastoma xenografts were stored at room temperature in RNAlater for 7 days or snap-frozen and stored at -70°C. RNA was then isolated from the tissue samples and analyzed by spectrophotometry, gel electrophoresis, RT-PCR analysis, and gene expression profiling. No major differences in RNA quality were observed.

This study showed that high quality RNA can be prepared from tumor tissue stored at room temperature in RNAlater. In cases when snap-freezing is not feasible, tumor tissue can be stored in RNAlater without damage to the RNA, thus simplifying large multi-institutional studies of RNA expression patterns for use as biological prognostic factors.

RNAlater Increases Yields of High Quality RNA for Microarray Analysis
Barrett, M. T., Glogovac, J. , Porter, P., Reid, B. J. and Rabinovitch, P. S. (1999) High yields of RNA and DNA suitable for array analysis from cell sorter purified epthelial cell and tissue populations. Nature Genetics 23: 32-33.

A recent paper in Nature Genetics reports that RNAlater simplifies the fixation of epithelial cells and tissues, and increases the yield of RNA and DNA obtained. MT Barrett and colleagues found that use of RNAlater allowed fluorescent staining and flow cytometric purification of whole epithelial cells. High yields of RNA and DNA were obtained from cell populations treated with RNAlater prior to cell sorting.

The authors of the paper were searching for a technology that would allow them to efficiently obtain high yields of high quality RNA and DNA from small cell samples. They were particularly interested in purifying RNA and DNA from neoplastic cells and tissues for genotyping and microarray analysis.

For their experiments, A549 lung adenocarcinoma cells and cells obtained from mastectomy samples were fixed in RNAlater. Single-cell suspensions were subsequently stained with fluorescent antibodies to cytokeratin and a nuclear counterstain. Cells were sorted by DNA content and cytokeratin staining intensity using a flow cytometer. RNA and DNA were isolated according to standard protocols and the integrity of RNA was assessed by real-time PCR assays.

The RNAlater-fixed cells showed good staining patterns with both the antibodies and DNA dyes. Real-time PCR analysis indicated the quality of the RNA obtained from RNAlater-treated cells and tissues was equivalent to control RNA and superior to RNA obtained from cells fixed in the traditional manner (EtOH/acetic acid fixation). Agarose gel analysis confirmed these results. Additionally DNA extracted from the RNAlater treated cells was isolated and used as a template for whole genome amplification, genotyping and sequencing. The researchers concluded that RNAlater can be used to obtain high yields of RNA and DNA suitable for expression analysis and genotyping.

Additional RNAlater citations