Historical Importance Of Ribonucleic Acids (RNA) Analysis In A Cell

In the cell, RNA is derived from the transcriptional product of deoxyribonucleic acids (DNA) and is further assisted through translation to create a protein. RNA applications include RT-PCR, cDNA synthesis, and northern blotting.

RNA Extraction

A great deal of evidence has suggested that deoxyribonucleic acid (DNA) is the carrier of information for the synthesis of proteins. Previous articles confirmed that an intermediate compound, ribonucleic acid (RNA), to be a template created from DNA to be used for protein synthesis (Brenner et al. 1961). On this confirmation, scientist have an opportunity to study the expression patterns of RNA. The efficacy of RNA extraction has been frequently enhanced over the years.

Pure RNA extraction is necessary for the analysis of gene expression in cells. Total isolation of RNA is essential although, RNA species are easily degradable. Many procedures exist in aiding in the pure isolation of RNA. A useful method containing guanidinium thiocyanate lysate and cesium chloride solution to cushion RNA based on the higher density species of RNA in comparison to DNA and proteins have been used in separations with high success (Chirgwin et al. 1979).

Procedures in the past have used hot phenol as their methodology of choice in extracting pure RNA (Chomczynski et al. 1987). The hot phenol method is a simplistic choice of extracting RNA in terms of speed and diverse range of the experiments one can conduct using the RNA isolated product. In essence one must work with haste and keep product in ice as much as possible. The theory behind RNA extraction is as follows: harvest cells, cell lysis, RNA binding, wash, and final elution

RNA gel blot- northern blot

An analysis of RNA can be done through northern blotting. Northern blotting is a supplementary advancement to the DNA analysis created by Ed Southern known as Southern blotting (Southern 1975). Unlike Southern blotting, northern blotting allows for the analysis of RNA separated through gel and is used to detect the presence and length of the RNA species. In 1977, a paper by Alwine et al.

First introduces RNA transfer techniques that differ from Southern blotting in that northern blot RNA species do not necessarily bind well to nitrocellulose. To enhance this method, the use of diazobenzyloxymethyl (DBM)-paper was first used to troubleshoot the issue and had high efficacy for single stranded nucleic acid transfer as well as high sensitivity to specific sequences labeled with 32P (Alwine et al. 1977). As previously described, total RNA can be extracted through acid and phenol/ guanidinium thiocyanate methods. Typically, RNA is separated in a denaturing agarose gel followed by the transfer of nucleic acid onto a paper, in which case Godt et al. used nitrocellulose and labeled [32P] dCTP probes were introduced for hybridization (Godt et al. 1997).

In 1977 Alwine et al. promoted a procedure in making a methyl mercuric hydroxide-agarose gel. However, the gel contains a powerful denaturing agent in methyl mercuric hydroxide that reacts with nitrogen atoms in the bases of nucleic acids (Alwine et al. 1977). The methodology behind this technique requires extra necessary steps to further remove the methyl mercuric hydroxide to avoid interactions within bases of the RNA. Today, gel electrophoresis separation is done by using agarose gel in conjunction with formaldehyde in the migration process to ensure inhibition of ribonucleases and to provide good separation of RNA species (Streit et al. 2009).

The separated RNA within the gel needs to be transferred onto a membrane for hybridization with labeled probes. The process of obtaining nucleic acids onto the filter does not differ from the Southern blot methodology. Following electrophoresis, the gel is immersed in ethidium bromide buffer that helps transfer the RNA molecules upwards toward the filter papers where the nucleic acids get trapped on a positively charged nylon membrane with a weight sitting on top

The transfer of nucleic acids from agarose gel is supported by the upward travel of buffer from the transfer buffer traveling towards the paper towels. The nucleic acids are joined with the nylon membrane and are radioactively labeled. Northern blots are diverse in that they can be probed with either oligonucleotides, RNA, and DNA. The labeling of 32P can be done with random or specific primers that can bind onto the RNA species which also allows for higher sensitivity for detection onto x-ray film during autoradiography (Streit et al. 2009). RNA in Situ Hybridization. In Situ hybridization of RNA can be defined as a method of locating specific transcripts within a tissue.

In 1968, the first experiments detecting amplified RNA in oocytes of Xenoponus were conducted with high success although, it was only ten years later that fluorescent tags were incorporated (Gall 1968). In 1977, Rudkin et al. introduced one of the first immunofluorescent assays involving DNA-RNA species. The experiment conducted involved a rabbit anti-hybrid antiserum that reacted to anti-rabbit immunoglobulin G tagged with rhodamine which allows for the detection on a specialized immunofluorescent module attached to a microscope (Rudkin et al. 1977). Rapidly, methods such as ISH and WISH became methods of choice to observe RNA and DNA species at a molecular level.

In Situ hybridization (ISH) is a powerful method used to study spatial template of expressed genes using a labeled DNA or RNA probe within tissue regions (Engler et al. 1998). ISH has many requirements including high resolution, selectivity and sensitivity (Engler et al. 1998). However, a new system in whole-mount in Situ hybridization (WISH) was developed using nonradioactive labeling probes. In comparison to ISH, WISH allows for the observation of transcripts in cells rather than tissue regions of plants or animals.

In 1989, Tautz et al. first developed a system of mRNA visualization in embryos of Drosophila melanogaster. The embryos in whole were hybridized with fragments from the coding region in a) hunchback b) Krüppel c) knirps d) fushi tarazu (Tautz et al. 1989). The technique required the immediate fixation of cells including all nucleic acid species. The use of paraformaldehyde allows for good condition of the fixated tissue with low amounts of cross-linked RNA allowing the nucleic acid species to be more accessible to probes (Engler et al. 1998). Depending on tissue type, the tissues may undergo pre-washing steps to further mount the cell onto glass plates. Upon washing, staining and addition of antibody-conjugate solution may be installed (Tautz et al. 1989).

According to a procedure written in 1997 by Tom McCreery, labeling of probes can be done in many ways according to the specific procedure one is using. The three procedures one can take for in Situ hybridizations are 5’ end labeling of oligonucleotides, 3’ end labeling of nucleotides, and RNA labeling by means of in vitro transcription. Due to high danger risk with the use of isotopic labeling, digoxigenin labeling became popular for its low biohazard risk. Radioisotopes also require special facilities and are highly unstable. The molecule digoxigenin is a steroid derived from Digitalis purpura a plant and can bind to a spacer by an ether bond which in case makes digoxigenin labeled nucleotides stable under alkali conditions as opposed to its competitor biotin.

Digoxigenin labeling may stay stable for a longer duration. Upon labeling RNA at the 3’ end with desired oligonucleotide of interest with digoxigenin tag, the tissue is incubated with an antibody against digoxigenin bound to an alkaline phosphatase (Chevalier at al. 1997). The digoxigenin antibody has a bound enzyme that will react when the appropriate substrate is added. A solution with 5-bromo-4-chloro-3-indolyphosphate/nitroblue tetrazolium with the appropriate substrate allows for blue-purple color intensity to be seen on the fixed tissue sample (Chevalier et al. 1997).

In conclusion, in Situ hybridization methodologies have greatly been enhance over the years and is an important way to observe expression and localization patterns of genes.

References:

Alwine, J. C., Kemp, D. J., & Stark, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences, 74(12), 5350–5354

Brenner, S., Jacob, F., & Meselson, M. (1961). An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis. Nature, 190(4776), 576–581

Chevalier, J., Yi, J., Michel, O., & Tang, X.-M. (1997). Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand? Journal of Histochemistry & Cytochemistry, 45(4), 481–491

Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156–159

Engler, J. D. A., Montagu, M. V., & Engler, G. (1998). Whole-Mount in Situ Hybridization in Plants. Arabidopsis Protocols, 373–384.