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Ultrapure water drives RNA technologies: the use of ultrapure water in RNA research using in situ hybridization ensures contamination-free, reliable results.

In situ hybridization (ISH) for localization of DNA/RNA hybrids in cytological preparations was first described in 1969 by Gall and Pardue. This method enables mRNA transcripts to be detected in tissue sections. Unlike expression analyses based on polymerase chain reactions, the exact localization of the target transcripts can be identified within the tissue.

ISH represents an alternative to immunohistochemical staining if no adequate antibodies are available, and is employed in diverse areas of research. This type of hybridization uses specific nucleic acid fragments (probes) that are complementary to the target sequence in order to detect specific transcripts. These probes may comprise DNA or RNA. Meanwhile, RNA probes are frequently utilized. Such probes are commonly labeled with the molecule digoxigenin (DIG), which normally occurs in the plant species Digitalis purpurea. DIG labeling enables the specifically bound probes in a tissue section to be visualized by enzyme-conjugated anti-DIG antibodies. For this purpose, after incubation of tissue sections with antibody, the appropriate substrate is pipetted onto the sections and converted by an immunoenzymatic reaction into a visible colorant (Figure 1). This method can be used to analyze the activity of specific genes for research projects or diagnostic procedures.

This article discusses the results of ISH that was carried out as part of a cancer research project. The analyzed skin samples were obtained from genetically modified mice. Based on targeted manipulation of the tumor suppressor gene Patched (Ptch) by homozygous knockout, the Ptch-knockout mice developed basal cell carcinomas. These skin tumors are those that most commonly occur in humans. They frequently exhibit increased activity of the signaling pathway that is regulated by Patched. Inactivation of this important component in this mouse model pathologically activates the signaling pathway. This results in an increased expression of the target gene Gli1--a transcription factor that activates a variety of other genes--in tumor cells, and expression of Gli1 can be detected using ISH.

Importance of ultrapure water

The use of pure RNase-free water, chemicals and materials in this sensitive detection method is extremely important to prevent degradation of the RNA probe. In the studies presented in this paper, the highest requirements were placed on the purity of the water by using the arium pro VF system from Sartorius.

The water quality produced in this manner is used for highly sensitive analyses involving ISH in human genetics laboratories, among other applications. The research results obtained are described in the following.

Materials and methods

Normally, diethyl pyrocarbonate (DEPC) is added to the buffers and solutions required in order to inactivate RNases, which is tantamount to overkill, or excessive prevention of secondary contamination, such as that resulting from impure chemicals or materials. This treatment generates additional costs and is time-consuming, hazardous to the health of laboratory staff and not effective for all solutions. In fact, DEPC is ineffective in removing secondary contaminants from solutions containing Tris. For this reason, the use of DEPC for decontamination should be avoided, whenever possible, and ultrapure water free of contaminants should be employed instead.

As experience has shown, all reagents used, as well as water for ISH experiments, must have consistently high quality; i.e., they must be free of biological contaminants, such as organisms, DNases, RNases, endotoxins, etc.

However, this is not always the case for tap water depending on its origin. For instance, depending on local groundwater quality, water may contain varying levels of biological contaminants and chemicals.

For these reasons, ISH can not be performed using untreated tap water, as this would entail unnecessary time and expense.

Experimental

Tissue samples were taken from tumorous skin tissue in mice and embedded in paraffin. The paraffin-embedded tissue samples were then sectioned using a microtome. Afterward, the paraffin-embedded sections were deparaffinized and rehydrated, and the tissue was permeabilized with proteinase K. To prevent unspecific binding because of differences in electric charge, the tissue samples were incubated with acetic acid anhydride. The Gli1-specific DIG-labeled RNA probe was incubated on the tissue sections overnight at 59 C. To remove any unspecifically bound probe, a stringent washing protocol was performed. In this procedure, the tissue sections were repeatedly washed at 63 C in a solution containing formamide and the unbound probes were removed by incubation with RNase A. A DIG-specific antibody (Roche) was used to detect the DIG-labeled probe. First, the sections were treated with I-Block (Tropix) to block any unspecific antibody binding sites. Then anti-DIG-antibody was added and the sections were incubated overnight at 4 C. On the following day, the sections were washed to remove unbound antibody. For detection of the probe, the enzyme alkaline phosphatase that was bound to the antibody was used. Therefore NBT/ BCIP substrate was applied to the sections. Alkaline phosphatase causes conversion of the substrate into a blue chromogenic product. The sections were examined under a light microscope, analyzed and photographed.

Results

Figure 2 shows the result of hematoxylin-eosin (HE) staining (2a) and ISH for the Gli1 transcript of a basal cell carcinoma. HE staining allows for the identification of tumor cells located below the epidermis in the dermis. With this staining method, the cell nuclei and cytoplasm are dyed purple-blue and pink, respectively. In contrast to this method, ISH enables the detection of a specific transcript, in this case, Gli1.

The results of ISH with the specific Gli1 probe are shown in Figure 2b. The specific probe, which is usually designated as antisense probe, is complementary to the mRNA of Gli1. A so-called sense probe (Figure 2c) is used as a negative control. It has the same sequence as the mRNA and can therefore not bind to the Gli1 transcript. As shown in Figure 2b, Gli1 is expressed in the tumor cells and not in the epidermis or dermis.

By contrast, tumor cells are not stained when the unspecific sense probe is used (Figure 2c). Based on these results, it can be assumed that the Patched signaling pathway exhibits strongly increased activity in tumor cells. Contamination with RNases from non-purified water or reagents would have led to degradation of the probes, and detection of Gli1 with the antisense probe would have been impossible.

Discussion

The ISH method introduced in this paper serves to localize transcripts within a tissue. Normally, the conventional labor- and cost-intensive ISH protocol takes several days and is performed in various ways in different laboratories. In principle, DNA probes can be used instead of RNA probes to detect mRNA transcripts. The advantages of DNA probes are higher stability compared with RNA and thus easier handling as they cannot be degraded by RNases. However, RNA-RNA hybrid duplexes are more stable than are RNA-DNA hybrid duplexes and, for lab staff experienced in handling RNA-RNA probes, are therefore preferable to RNA-DNA. To prevent RNases from degrading the RNA probe, a careful work method is necessary, and all buffers and solutions should be prepared with RNAase-free ultrapure water as far as possible.

Ultrapure water produced using Sartorius' arium pro system is characterized by the consistently high quality of all water specifications required, such as conductivity/resistivity, TOC, RNases/DNases and endotoxins. Particularly in the case of endotoxin levels, it was recently demonstrated that ultrapure water produced by arium pro VF exhibits exceptionally low concentrations of <0.001 EU/mL and is thus well below the typical limits.

The arium pro VF system was designed to produce ultrapure water from pretreated drinking water by removing contaminants that are still present in this potable water feed. Production of ultrapure water requires continuous recirculation and a constant water flow rate, which is achieved using a built-in pump system with controlled pressure. The conductivity of the water is measured at the feed water inlet and directly at the downstream port (product water outlet).

The arium pro VF system used in this study works with two different cartridges. These are filled with a special active carbon adsorber and mixed-bed ion exchange resins in order to deliver ultrapure water with a low TOC content. In addition, the system has an integrated UV lamp that has a bactericidal and oxidizing effect at wavelengths of 185 and 254 nm, respectively.

Moreover, the ultrapure water system has a built-in ultrafilter module used as a crossflow filter. The ultrafilter membrane incorporated in this filter retains colloids, microorganisms, endotoxins, RNA and DNA and removes RNases, which is essential in order to perform ISH. A 0.2 pm final filter installed at the water outlet serves to remove particulates and bacteria during dispensing of the ultrapure water produced. The process that the unit employs to purify water is depicted in Figure 3.

Literature:

1. Gall, J.G., Pardue M.L.: Formation and detection of RNA-DNA hybrid molecules in cytological preparations, Proc. Natl. Acad. Sci., USA, 63, No. 2, 378-383 (1969).

2. Wilcox, J.N.: Overview of in situ hybridisation methodology, Workshop--Visualizing Neoplasia. Year 2000 Meeting of the Histochemical Society, Emory University, Winship Cancer Institute, Division of Hematology/Oncology, Atlanta, GA, USA (2000).

3. Zibat, A., et al.: Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts, Carcinogenesis, 30 No. 6, 918-926 (2009).

4. Grundwasser in Deutschland: Bundesministerium fur Umwelt, Naturschutz und Reaktorsicherheit (BMU), 1. Auflage, Stand August (2008).

5. Rund um das Trinkwasser: Umweltbundesamt, (2011) 2. Auflage, Redaktionschluss November (2010).

6. Sugimoto, N., et al.: Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes, Biochemistry 34, 11211-11216 (1995).

7. Schmidt, K., and Herbig, E.: "Weniger ist mehr--Quantitative Endotoxinbestimmung von Reinstwasser", Laborpraxis 5, 36.Jhg. (2012).

by Frauke Nitzki, Institute of Human Genetics, Germany, and Elmar Herbig, The Sartorius Group, Goettingen, Germany
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Author:Nitzki, Frauke; Herbig, Elmar
Publication:Chromatography Techniques
Geographic Code:1USA
Date:Sep 1, 2013
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