In situ hybridization histochemistry with synthetic oligonucleotides: Strategies and methods

Abstract

In situ hybridization histochemistry is a useful method for localizing specific mRNA and studying the regulation of gene expression in an anatomical context. Previously, classical recombinant DNA and microbiological techniques have been required to identify and nick-translate the cloned DNAs necessary for in situ hybridization experiments. These requirements can be circumvented by the use of synthetic oligonucleotides complementary to the mRNA of interest. Compared to cloned cDNA probes, oligonucleotides are easy to manufacture, penetrate tissue much more easily, can be made to correspond to a sequence at any point in a known cDNA structure, and allow for the design of more precise controls for in situ studies. We describe a number of considerations in oligonucleotide probe design, including unique probe design from cDNA sequences and mixed probe design from protein primary structure data. The issues of species specificity, G-C content, probe length, tissue-specific mRNA expression, repeated sequences, non-coding region specific probes, and gene family homologies are discussed in an in situ hybridization context. Alternative strategies for mixed probe design are also considered. Information on the synthesis, purification, and sequence confirmation of oligonucleotides is then presented, followed by methods for labeling and using these probes for in situ hybridization histochemistry. The special considerations of specificity controls are addressed, including combined in situ hybridization histochemistry and immunocytochemistry, competition studies, the use of multiple hybridization probes, Tm studies, and Northern analysis of extracted RNA. The current and future directions of research with this technique are considered, with emphasis on the need to improve quantitation in order to facilitate the study of gene expression and regulation at the single cell level.

Collagen gene expression during development of avian synovial joints: Transient expression of types II and XI collagen genes in the joint capsule

“…compound and cryosectioned at 14 pm. Frozen sections were prepared for in situ hybridization as described by Lewis and associates, (Lewis et al, 1985) with modification as described by Sandell and associates (Sandell et al, 1991). Exposure times were 3 days for type I collagen and 7 days for type IIA, IIB, and XI collagens.…”

Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non‐cartilaginous tissues during early mouse development

“…compound and cryosectioned at 16 pm. Frozen sections were prepared for in situ hybridization as described by Lewis and associates, (Lewis et al, 1985) with modification as described by SandJl and associates (Sandell et al, 1991). In brief, slides with sections of tissue were warmed to room temperature, post-fixed in 4% paraformaldehyde, treated with acetic anhydride (0.25% in 0.1 M triethanolamine), dehydrated, delipidated, and air-dried.…”

Temporal expression of growth factors and matrix molecules in healing tendon lesions

“…[3SS]UTP-labeled sense and anti-sense riboprobes (NEN Life Science Products, Inc., Boston, MA) were synthesized, and applied to serial sections on processed slides [14,21]. Probes corresponded to the 5' most 254 and 287 nucleotides of the mRNA for collagen types I and 111, respectively, the 270 nucleotides encoding exons 1 to 3 of the IGF-IA precursor, and the 250 nucleotides encoding exons 6 and 7 of TGF-PI.…”