Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes.
In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.
Darnell
Diana K
DK
Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724, USA.
Stanislaw
Stacey
S
Kaur
Simran
S
Antin
Parker B
PB
D017398:Alternative Splicing; D000818:Animals; D002642:Chick Embryo / Q000737:chemistry*; D064211:Connectin; D005821:Genetic Techniques; D017403:In Situ Hybridization / Q000379:methods*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*
D064211:Connectin; D009124:Muscle Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; C477371:locked nucleic acid