In situ hybridization protocol

Tata, AM. 2001. An in situ hybridization protocol to detect rare mRNA expressed in neural tissue using biotin-labelled oligonucleotide probes. Brain Res Prot 6 178-184. 

As described in Chapter 46, virtually all in situ hybridization protocols entail the following major steps ... 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Experimental samples are mainly derived from tissue culture cells, laboratory animals, or human tissues collected from hospitals after surgical biopsies and autopsies. With human and animal tissue specimens, it is important to arrest metabolic processes within 5-10 min of collection in order to preserve mRNAs from degradation by internal enzymatic reactions (26,27). Most hospitals use 10% buffered formalin as a tissue fixative. Subsequently, each tissue slice is trapped in a paraffin block. Series of 4-5-pm-thick sections are cut and mounted on silanated slides. Formalin-fixed archival tissues have been successfully used in in situ PCR and in situ hybridization protocols (28-32). However, the procedure for RNA protection is not always followed. It is often difficult to alter or control the routine procedures of hospitals for the required protection of mRNAs in surgically removed human tissues. 

Nuovo, G. J. (1997) PCR in situ hybridization protocols and applications, 3rd ed. Lippincott-Raven, Philadelphia, PA. 

Jorgensen S, Baker A, MoUer S et al (2010) Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes. Methods 52 375-381... 

ISH is versatile and can be used for the detection of viral targets and for a number of other diagnostic tests including in the assessment of B-cell clonality by light chain restriction. Recent developments that have led to widespread utility of ISH include the development of nonfluorescent chromophore in situ hybridization protocols that permit the ISH analysis of a variety of targets using the light microscope. ... 

In Situ Hybridization Protocols, edited by K. H, Andy Ckoo, 1994... 

Standard in situ hybridization protocols, involving hybridizations in formamide at 65 C overnight, have been reported to quench GFP s fluorescence (Brand 1999 Hazelrigg lab observations). 

Preparation of polytene chromosomes for in situ hybridization Protocol 25... 

Kiyama, H., Emson, P.C., and Tokyama, M. (1992) In situ hybridization histochemistry using alkaline phosphatase-labeled oligodeoxynucleotide probe. In Methods in Molecular Biology, Protocols in Molecular Neurobiology, (A. Longstaff, and R Revest, eds.), Vol. 13, pp. 167-179. Humana Press, Totowa, New Jersey. 

Armed with the common techniques of molecular biology and immu-nocytochemistry, an investigator is in a good position to apply in situ hybridization to EM for localization of nucleic acids at the ultrastructural level. McFadden (9) has a review on such use of in situ hybridization techniques. In the review, McFadden has included details of some of his laboratory protocols needed in in situ hybridization from fixation and labeling to probe labeling to the hybridization steps for localization of specific RNAs at the EM level. His protocol for hybridization is outlined below ... 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

This protocol is based on one used in the in situ hybridization course directed by Jan Kuzava Blancato and held at the Center for Advanced Training in Cell and Molecular Biology. Julie Brent and Dwayne Dexter contributed to development and support services. 

Lichter, P. and Reid, T. (1994) Molecular analysis of chromosome aberrations in situ hybridization, in Chromosome Analysis Protocols (Gosden, J. R., ed.), Humana, Totowa, NJ, pp. 449 78. 

Fig. 4. In situ hybridization technique. Part of an erythroid precursor cell infected with the human parvovirus B19 and probed for viral DNA. The BI9 nucleic acid is located within the centra electron lucent area of the nucleus (N) and also at nuclear pores (arrowheads). Ch, chromatin M, mitochondrion. A three-step detection protocol was used (see ref. 5 for details). Sheep antidigoxigenin followed by rabbit antisheep Ig and then goat antirabbit Ig conjugated to 10-nm gold. Bar is 0.5 pm.

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