Retrieval of mRNA from paraffin-embedded human infant brain tissue for non-radioactive in situ hybridization using oligonucleotides.
In situ hybridization (ISH) is used to examine the spatiotemporal distribution of gene expression in a range of tissues. Neuroscience research in human brain tissue requires techniques that can be used in formalin fixed and paraffin-embedded tissue rather than frozen tissue which is recommended, but difficult to obtain. This study presents a method for non-radioactive (DIG) ISH for detecting NR1 gene expression, in human infant brain tissue. We compared three pre-treatment effects, protease digestion, autoclaving (in citrate and Tris/EDTA buffer) and microwaving (in citrate and Tris/EDTA buffer). Tissue had been fixed in formalin for 2-12 weeks. Results were compared for the hybridization and background signal intensities, and tissue morphology. We found that optimum results were obtained using 12-min microwave pre-treatment in Tris/EDTA buffer. This method produced optimum signal to background ratio in infant and adult tissue, preserved tissue morphology, and was suitable for use across a broad range of fixation times.
Relf
Bronwyn L
BL
Department of Medicine, Room 206, Blackburn Building, DO6, The University of Sydney, NSW 2006, Sydney, Australia.
Machaalani
Rita
R
Waters
Karen A
KA
D000368:Aged; D001921:Brain / Q000378:metabolism*; D002021:Buffers; D006801:Humans; D007158:Immunologic Techniques; D017403:In Situ Hybridization; D007223:Infant; D008875:Middle Aged; D015345:Oligonucleotide Probes; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000302:isolation & purification*
D002021:Buffers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger