Korean J Phys Anthropol.  2010 Jun;23(2):87-96.

Detection of alphaB-crystallin mRNA using Single-stranded DNA Probe in Oligodendrocytes of the Developing Chick Retina

• Kim JY
• Sohn HJ
• Seo JH

Abstract

In situ hybridization (ISH) using single-stranded DNA probe (ssDNA probe) is a useful method for observing the specific transcripts in cells, since it is convenient to prepare probe which is specific and sensitive. In this study, ssDNA probe for detection of alphaB-crystallin (aBC) mRNA, transcript of a heat shock protein, was prepared and aBC mRNA-expressed cells were spatiotemporally observed in the retina of the developing chick embryos. Single-stranded antisense probe produced by reverse transcription and polymerase chain reaction was identified as a specific probe for aBC mRNA in comparison to negative control using sense probe and immunohistochemistry for aBC protein. In the ISH experiment, aBC mRNA was expressed only in the peripapillary glial cells which are a specific cell type located in the avian retina adjacent to the optic nerve at E12 and E14 retinas. At E16, a small number of aBC mRNA-expressed cells were identified in the nerve fiber layer (NFL) of the retina. At E18, aBC mRNA-expressed cells were observed in the ganglion cell layer (GCL) as well as the NFL. At E20, the number of aBC mRNA-expressed cells was increased in the GCL and the NFL. Based on the same localization of nkx2.2 immunoreactive cells and aBC mRNA-expressed cells, aBC mRNA-expressed cells were identified as oligodendrocytes. These results indicate that ssDNA probe for aBC mRNA detection is very useful tool for oligodendrocyte research such as distribution, migration and differentiation of the cells.

Keyword

In situ hybridization; Single-stranded DNA probe; alphaB-crystallin; Retina

MeSH Terms

DNA, Single-Stranded
Ganglion Cysts
Heat-Shock Proteins
Immunohistochemistry
In Situ Hybridization
Nerve Fibers
Neuroglia
Oligodendroglia
Optic Nerve
Polymerase Chain Reaction
Retina
Reverse Transcription
RNA, Messenger
DNA, Single-Stranded
Heat-Shock Proteins
RNA, Messenger