PMC

Experimental overview of the SCN method and ISH. The AFM probe was inserted into a cell to take mRNAs, and then analyzed with RT-PCR, followed by quantitative PCR. The same cell was fixed by 4%folmaldehyde/PBS and subjected to ISH.

ISH result in higher resolution. Black color indicates high intensity of ISH. As black becomes white, the intensity of ISH decreases. The numbers of each figure are the β-actin mRNA quantity detected by the SCN method. Scale bar is 1 μm.

Relation between ISH intensity and the distance from nuclear membrane. The average of the ISH intensity was calculated according to the distance from the nuclear membrane. The detection probability of β-actin mRNA with the SCN method changed according to the distance from the nuclear membrane. As the distance from the nuclear membrane became greater, fewer positive results were obtained with the SCN method.

The results of the SCN method and ISH. (a-c) Each square indicates the region of the AFM probe insertion. Numbers in lower panels indicate β-actin mRNA quantities detected by the SCN method, and dark intracellular staining indicates distribution of β-actin mRNA detected by ISH. (d) Negative control using sense RNA probe. Scale bar is 50 μm.