In Situ Detection of Intracellular Messenger RNA and MicroRNA
Both messenger RNA (mRNA) and microRNA (miRNA) are emerging biomarker candidates for early disease diagnosis and prognosis and also therapy targeting. The detection of mRNA/miRNA abundance is significant for deciphering cellular signalling pathways, understanding cellular behaviour and predicting the malignant progression of tumours. Conventional techniques for RNA analysis such as northern blotting, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and DNA microarrays rely on the fixation or lysis of cells. However, these techniques are labour intensive and time consuming. More importantly, the information about the dynamics of RNA inside cells and cell-to-cell heterogeneity is often lost in such cases owing to ensemble averaging. The accurate analysis of the abundance of miRNA in real time in living cells is needed for monitoring the amount and localization of miRNA. However, monitoring of intracellular mRNA and miRNA levels in living cells remains a great challenge owing to the low gene probe transfection efficiency and the low abundance of mRNA and miRNA. This chapter first introduces the biofunction of mRNA and miRNA, then various gene probe vectors are summarized and the advanced techniques involved in intracellular mRNA and miRNA detection are discussed. Perspectives and challenges are also presented.