In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples.
High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is approximately 7 d, depending on the tissue used.
Hejátko
Jan
J
Masaryk University, Department of Functional Genomics and Proteomics, Laboratory of Molecular Plant Physiology, Kamenice 5, 625 00 Brno, Czech Republic.
Blilou
Ikram
I
Brewer
Philip B
PB
Friml
Jirí
J
Scheres
Ben
B
Benková
Eva
E
D017360:Arabidopsis / Q000737:chemistry*; D018506:Gene Expression Regulation, Plant; D017403:In Situ Hybridization / Q000379:methods*; D016372:RNA, Antisense / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000032:analysis*; D016707:Tissue Fixation
D016372:RNA, Antisense; D012333:RNA, Messenger