mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial β-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for β-actin mRNA. In the SCN method, quantity of β-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of β-actin mRNA. We showed that intensity of ISH is higher; quantity of β-actin mRNA detected by the SCN method increased more.In this study, we compare the SCN method with the ISH. We examined β-actin mRNA expression in single cells using both methods. We picked up β-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids, and it has attributed significantly to the advancement of the study of gene expression in cells and tissues. ISH was invented by two groups in 1969 [1,2]. Around that time, only radioisotope (RI) was available to label nucleic acids. But nowadays, non-RI ISH can be preformed based on synthesis of nucleotides containing certain functional groups and synthesis of a modified oligonucleotide by Digoxigenin (DIG) system [3-6]. Its primary advantage over the Northern blot and reverse transcription polymerase chain reaction (RT-PCR) is its ability to detect localization of specific mRNA to a particular cell or a particular region in a cell. So ISH are applied for bacteria, culture cells, tissue section and whole mount embryo [7-11]. However, ISH cannot examine time-lapse change of identical cells because the cells have to be fixed.We reported a single cell nanoprobe (SCN) method to examine mRNA expression without killing cells in a previous repor