Preserving Macromolecules in the Field - Research Proposal Example

Research Proposal: Preserving Specimens Client Inserts His/her Name Client Inserts Grade Course Client Inserts Tutor’s Name 28/10/2011 Research Question: How effective is Novel zinc-based fixative (Z7) as a cheap alternative media for preserving specimens (macromolecules) in the field? Hypothesis: Novel zinc-based fixative (Z7) is a cheap effective alternative media for preserving specimens (macromolecules) in the field. Rationale: Macromolecules in and around the cells determine the structure of a tissue. An essential part of all histological and cytological techniques is preservation of cells and tissues as they naturally occur. This is accomplished by immersing tissue blocks, sections or smears in a fixative fluid or merely drying (Karp 2009). Most biologists are interested in the three principal macromolecules (DNA, RNA and proteins). These have traditionally been preserved by snap freezing in liquid nitrogen followed by storage at -80oC, making the tissue suitable for future genetic and/or quantitative analysis. It is however unlikely that an investigator will have accesses to a -80oC freezer, never mind liquid nitrogen when in the field. Although alternative preservation methods to freezing exist, e.g. storing the tissue in ethanol, this is usually at the expense of the other macromolecules. This has led several manufactures to produce “wonder” media, e.g. RNAlater, which can be used to preserve all three macromolecules in the field at relatively high (4oC) temperatures. The only problem is that such media, unsurprisingly, is expensive. This has created the need to come up with a suitable cost effective alternative media for preserving materials in the field: Novel zinc-based fixative (NZBF) (Z7). There has been a call for development of a reliable, cost effective and non-toxic fixative that meets the needs of the contemporary molecular pathobiology. Previously used macromolecules preservation techniques have been found to be limiting or rather ineffective in one way or another (Buchwalow 2010). Snap freezing for example has been the best way to preserve specimens traditionally. Snap freezing is performed on a pre-cooled cool-Rack which ensures fast heat transfer. This method provides excellent specimen integrity and a wide array of options for tissue analysis, including extraction of proteins, DNA and RNA for use in research and diagnostics. However, the process has some drawbacks due to the formation of ice crystals within in the tissue, the compartmentalization within the cell will be disrupted and thawing of the tissue will turn it into a reaction tube full of RNA degrading activities (RNA Quality Control). The process is also limited by the fact that it is possibly hard that an investigator in the field will have access to a -80oC freezer. Freezers are not only expensive but also require to be powered which is hectic while in the field. Furthermore, normal freezers operate at -18oC and those that cool up-80oC to -80oC are mostly available in research institutions. The cost of liquid nitrogen further makes the process costly. Neutral Buffered Formalin (NBF) has also been among the widely used fixative in cytology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens (Wester et al. 2003). Precipitant fixatives like methanol, ethanol, acetone and acetic acid denature proteins by breaking the hydrophobic bonds that make up the tertiary structure of protein molecules yet preserve secondary structure for immunohistochemistry (ICH). Despite the number of fixatives available, however problems still remain for many of them including toxicity, expense, the need for rapid fixation systems and the need to employ denaturants and low melting point wax for embedding (Lykidis et al. 2007). The important aspect of this study is to show that NZBF can be used as a suitable cost effective alternative media of preserving specimens. Lykidis et al. (2007) in their study shows that NZBF containing zinc trifluoroacetate, zinc chloride and calcium acetate are significantly better than NBF and other formerly used fixatives for DNA, RNA, and protein preservation. Aims and Objectives: The aim of the present study is to show that NZBF (Z7) can be used as a cheap alternative media for preserving specimens (macromolecules) in the field. The objectives of the study: 1. To determine the effects of NZBF on tissue morphology. 2. To determine the effects of NZBF on the quality of genomic DNA. 3. To determine the effects of NZBF on preservation of proteins. Literature Review DNA-based methods have revolutionized in most areas of biological research during the last decade. While DNA isolation techniques are constantly being improved, the impact and importance of specimen preservation are greatly being underestimated (Nowacek J. M.). Methods of preserving biological specimens have become more important due to recent advances in molecular biological analysis (Takahashi et al. 1995). An appropriate fixation of biological specimens will have a major impact on the quality of specimens to be preserved and the level of analysis to be made. Fixation in this case can be defined as a series of complex chemical modifications of the macromolecules present in tissues and cells, to preserve structural and functional components as possible t the living state while inhibiting autolysis, bacterial and fungal decay (Lykidis et al. 2007). Maintaining the specimen morphology is the major prerequisite for good immunostaining. If the origin is garbage, do not expect wonderful results (Buchwalow 2010). The structure of the tissue is determined by the shapes and sizes of macromolecules in and around cells. The principle macromolecules inside a cell are proteins and macromolecules. The fixatives employed prevent autolysis by inactivating lysosomal enzymes, and they stabilize the fine structure, both inside and between the cells, by making macromolecules resistant to dissolution by water and other liquids (Nowacek J. M.). The goals of fixation are to preserve the structure of cells with minimum alteration from the living state with regard to volume morphology, and spatial relationships of organelles and macromolecules, minimum loss of tissue constituents, and protection of specimen against subsequent treatments including rinsing, dehydration, staining, vacuum, and exposure to electron beam (Rebecchi et al. 2005). Ideally, the aim of a desirable fixation method is satisfactory preservation of cells as a whole and not the best preservation of only a part of it (Hayat 2000). Shi and Taylor (2010) argue that microscopic evaluation of a tissue section taken from a specimen remains the gold standard for determining its macromolecular makeup. Analysis of genomic DNA, transcribed genes, and expressed proteins add important information to the histological features detected in the microscope (Wester et al. 2003). Short DNA and RNA sequences can be retrieved from conventionally fixed pathology material, but good, long-term preservation of intact nucleic acids and protein integrity is necessary to meet the increasing number of molecular diagnostic and research techniques which are becoming available. The type and length of fixation determine the degree of preservation of intact nucleic acids in tissues (Lykidis et al. 2007). According to Matsuo et al. (2002), there may be limited access to materials and equipment necessary while in the field, so preliminary preservation with more simple methods may be necessary before final preparation as a permanent collection specimen. Precipitant fixatives have been found to denature proteins in the specimens hence flagged as inappropriate for preservation. Other compounds like the commercially available HOPE (HEPES- Glutamic acid buffer mediated Organic solvent Protection Effect) preserves DNA and RNA suitable for polymerase chain reaction (PCR) and reverse-transcription (RT) -PCR and the reversible cross-linker dithio-bis [succinimidyl propionate] (DSP) for immunostaining, micro-dissection and expression profiling (Lykidis et al. 2007). Traditionally, Fixation in 10% buffered formalin followed by paraffin embedding has been used for specimen preservation. However, use of formalin as a fixative and preservative is facing increasing criticism because of toxicity and environmental pollution (Olsen et al. 2009). Moreover, the analysis of mRNA and DNA from formalin fixed paraffin embedded tissue has been realised to be problematic. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. It is essential to develop alternate methods for better preservation of macromolecules that will preserve the tissue microanatomy also. There have been numerous attempts to find formalin substitutes that are more rational, safe and reliable (Shankar & Pal 1999). Formaldehyde is one of the most popular fixatives. This is due to its low cost, easy to prepare and preservation of tissue morphology. However, formaldehyde fixation causes variable yet reversible loss of immunoreactivity by masking or damaging some of the antigens binding sites (Shankar & Pal 1999). The deleterious effects on DNA and proteins caused by formaldehyde fixation are well known. Initially, there is a reversible and rapid hydroxymethylation of amino and imino groups of the nucleic acid bases, followed by a slow reaction resulting in methylene bridge formation between bases (Wester et al. 2003). Formaldehyde also forms cross-linking of proteins to DNA, and the degree of this process depends on the duration of fixation (Shi, Key & Kalra 1991). A new universal molecular fixative (UMFIX) for preservation of macromolecules in paraffin-embedded tissue has been tested and established that it can preserve the morphology of macromolecules but still problems of toxicity and expense remain (Vincek et al. 2005). UMFIX is a methanol, polyethylene glycol based fixative and usually joined with microwave assisted rapid tissue processing. It is very useful for small biopsies to amplify small ampicons by RT-PCR. Tissues can be fixed in room temperature with UMFIX but the disadvantage mentioned above and the fact that it is assisted by microwaves makes it hard to be usable working outback (Shankar & Pal 1999). A zinc based fixative (ZBF) (zinc acetate, zinc chloride and calcium chloride in Tris buffer) has been documented to be superior for DNA and protein extraction analysis in a broad spectrum of tissue and does not require heat pre-treatment for antigen retrieval. Of late, modified NZBF (Z7) in 0.1% M Tris-HCL, PH 6.4-6.7 is reliable, cost effective and non-toxic as compared to other fixatives and preservation methods. The veracity of DNA, RNA and protein appeared excellent, therefore molecular analysis on NZBF (Z7) fixed paraffin embedded tissue samples is better to the other fixatives fixed tissues (Shankar & Pal 1999). In other studies zinc-fixed, paraffin-embedded tissues provided superior morphology and improved immunostaining. Zinc compounds are non-toxic and inexpensive, non-carcinogenic and are not temperature sensitive. Vincek et al. (2003) put it that of the other elements in group 12 of the periodic table (Zn, Cd, Hg), zinc is the least toxic. Zinc sulphate has been used for more than a century in caustic creams and eye drops, which lowers the swelling of irritated surfaces by clotting extravagated plasma proteins (Garcia et al. 1993). The first zinc-formalin fixative was probably the one published by P. A. Fish in 1895, only three years after the discovery that formaldehyde preserved tissue architecture very well. In recent decades several intelligently formulated fixatives have been developed. A few are entirely coagulant, with Zn2+ as the only ingredient likely to immobilize proteins. Most zinc-containing fixatives also contain formaldehyde, making them comparable to B5 and other mercury-containing solutions (Nowacek J. M.). NZBF has proved beneficial in histochemical analyses performed on intact tissues but has not been exploited in procedures that focus on quantitative analysis of individual cells (Stanta 2011). As an alternative to formaldehyde-based fixation, NZBF, not containing any cross-linking agents, is also effective in histopathology procedures. Studies have revealed that RNA, DNA, and proteins are preserved well again in NZBF than in NBF. Furthermore, it was shown that the morphological preservation was similar to aldehyde-based fixation (Jensen et al. 2010). Of late, studies show that enzymes maintain their activity in NZBF-fixed tissue permitting analysis of enzymatic activity in tissue sections. Nevertheless, NZBF has not been widely used in practice pathology where NBF is still the favoured fixative (Paavilainen et al. 2009). NZBF has recently been greatly used in the preparation of tissue samples for immunohistochemical analysis. This is because NZBF-fixed tissues are well preserved, exhibit improved immunolabeling of sensitive epitopes and maintains intact DNA and RNA to a higher extent compared to other fixatives. Also, ZNBF is nonhazardous, easy to prepare and inexpensive. The only disadvantage of NZBF is incomplete penetration of tissues as compared to NBF but form the above mentioned advantages it is the best alternative fixative at the moment (Jensen et al. 2010). Methodology The research will employ experimental method design. Experimental research is usually aimed at investigating the possible causes and effects relationship. The independent variable is presumed to be the “cause” while the dependent variables are presumed to be “effects” (Gliner, Morgan & Leech 2009). Sometimes they are aimed at explaining certain phenomenon. This is done by manipulating an independent variable thereby influencing the other variables in the experimental group. In an experiment, researchers are mostly concerned about the performance of subjects in the experimental group. The manipulation of an independent variable enables the researcher to see if the treatment makes a difference on the subjects (Weiten 2010). In this research, the independent variable to be manipulated is the fixative which in our case is the NZBF. The experimental method design is regarded as the most scientific of all the methods research design has to offer. Some refer to it as the “method of choice”. While other research method designs face the problem of lack of control over the situation, experimental method design is a way of overcoming this problem (Goodwin 2009). Tissue sampling Tissues from specimens collected in the field will be sampled randomly for the experiment. Tissues from various organs obtained from the collected and sampled specimens will be used for the experiment. These include: Tissue Organ Skin Liver Colon Lung Stomach Kidney Different tissues will be used so as to assess the physical properties of the fixatives: penetration of the fixatives in different types of tissues. Four pieces of about 3 mm thick will be cut from each tissue for every experiment and repeated twice. The control experiment involved snap freezing tissue samples in order to determine their DNA and RNA quality. In each experiment, two tissue pieces will be prepared both for NZBF and snap freezing separately. The first set of tissues will be preserved using each method for 24 hrs while the other set will be preserved for one week then analysis made. Tissues will be processed according to standard procedures after NZBF- fixation and then paraffin embedded. The paraffin blocks serve as donor block for the construction of tissue array. Snap freezing will be done at -80oC and according to the rest of the procedures standard. Tissue sections will be cut at 4-m thickness and floated on distilled water. The cut sections will then be picked up on charged slides and air dried for one hour. Histologic staining and IHC staining will then be done according to the procedures for paraffin-embedded tissue sections. For IHC, tissue sections will be immunostained using Dako Cytomation Animal research Kit (ARK) - biotinylated antibody method and no antigen retrieval was performed. PCR and RT-PCR will be performed on genomic DNA to amplify two universally expressed genes, GAPDH- -actin and Trefoil factor 2 genes. DNA and RNA extraction DNA and RNA will be extracted using any commercially available kits from fixed paraffin-embedded tissue and according to the manufacturer’s protocol. This was also done to the snap-frozen tissues. Slides containing DNA and RNA will be observed, evaluated and scored under the microscope. A comparison will be made between the NZBF and the snap-frozen results. Appendix A [NZBF (Z7) Standards] 17.15mM zinc trifluoroacetate 0.5% zinc chloride 0.05% calcium acetate 6.4-6.7 PH Appendix B (Research Schedule) Duration (Weeks) 1st Week 2nd Week 3rd Week 5th Week 6th Week Activity Research proposal writing Specimen collection and experiments Experiments Results analysis Research report writing List of References Buchwalow, I. B., 2010. Immunohistochemistry: Basics and Methods. London: Springer. Garcia, L. S., Shimizu, R. Y., Shum, A., and Bruckner, D. A., 1993. Evaluation of intestinal protozoan morphology in polyvinyl alcohol preservative: comparison of zinc sulfate- and mercuric chloride-based compounds for use in Schaudinn's fixative, Journal of Clinical Microbiology, [Online]. Available at: [Accessed 21 October 2011]. Gliner, J. A., Morgan, G. A., and Leech, N. L., 2009. Research methods in applied settings: an integrated approach to design and analysis. 2nd ed. London: Routledge. Goodwin, J. C., 2009. Research in Psychology: Methods and Design. 6th ed. New Jersey: John Wiley and Sons. Hayat, M.A., 2000. Principles and techniques of electron microscopy: biological applications. 4th ed. Cambridge: Cambridge University Press. Jensen, U. B., Owens, D. M., Pedersen, S., and Christensen, R., 2010. Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes, Cytometry Part A, [Online]. Available at: < http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20914/full> [Accessed 21 October 2011]. Karp, G., 2009. Cell and Molecular Biology: Concepts and Experiments. 6th ed. New Jersey: John Wiley and Sons. Lykidis, D., Noorden, S. V., Armstrong, A., Dene, B. S., Li, J., Zhuang, Z., and Stamp, G. W. H., 2007. Novel zinc-based fixative for high quality DNA, RNA and protein analysis, Nucleic Acid Research, [Online]. 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Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections, Journal of Histochemistry and Cytochemistry, [Online]. Available at: [Accessed 22 October 2011]. Stanta, G., 2011. Guidelines for Molecular Analysis in Archive Tissues. London: Springer. Takahashi, R., Matsuo, S., Okuyama, T., and Sugiyama, T., 1995. Degradation of macromolecules during preservation of lyophilized pathological tissues, PubMed.gov, [Online]. Available at: [Accessed 21 October 2011]. Vincek, V., Nassiri, M., Block, N., Welsh, C. F., Nadji, M., and Morales, A. R., 2005. Methodology for Preservation of High Molecular-Weight RNA in Paraffin-Embedded Tissue: Application for Laser-Capture Microdissection, Diagnostic Molecular Pathology, [Online]. Available at: [Accessed 22 October 2011]. Vincek, V., Nassiri, M., Nadji, M., and Morales, A. R., 2003. A Tissue Fixative that Protects Macromolecules (DNA, RNA, and Protein) and Histomorphology in Clinical Samples, Laboratory investigation, [Online]. Available at: [Accessed 21 October 2011]. Weiten, W., 2010. Psychology: Themes and Variations. 8th ed. Stamford: Cengage Learning. Wester, K., Asplund, A., Bäckvall, H., Micke, P., Derveniece, A., Hartmane, I., Malmström P. U., and Pontén, F., 2003. Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues, Laboratory investigation, [Online]. Available at: [Accessed 19 October 2011]. Wester, K., Asplund, A., Bäckvall, H., Micke, P., Derveniece, A., Hartmane, I., Malmström P. U., and Pontén, F., 2003. Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues, Laboratory investigation, [Online]. Available at: [Accessed 19 October 2011]. Read More