Tissue specific RNA in-situ hybridization is a critical technique required for botanists to examine exact gene expression domain within plant tissues. This powerful technique provides a way to examine the spatial and temporal patterns of gene expression of plants and animals in developmental stages content and at tissue cellular level. However, this technique has been largely hampered by difficulties of obtaining reliable protocols for non-model organism. Here we offer our lab experiences on how to optimize in-situ hybridization process among various plant species. This step-by-step guide however should not be regarded as the only solution to resolve the difficulties of RNA tissue specific RNA in-situ hybridization. Instead, the key optimization steps including tissue fixation time, proteinase/pronase treatment, probe length/content and hybridization time are priority issues to be tested first. We further summarized some recent advance of literature on alternative ways to conduct in-situ hybridization process.