== (A) Schematic representation of the experimental set-up used in mRNA extraction (not to a scale). co-purified with RNA may introduce unwanted products during downstream applications (e.g. contamination with genomic DNA could introduce an unwanted background signal in a RT-qPCR assay)[1]. In addition, rRNA that constitutes more than 80% of total RNA may compete for reagent in the cDNA preparation reaction [1,4]. There is a need for clean, highly sensitive techniques for non-destructive quantitative analysis of mRNA levels within living cells. Atomic Pressure Microscope (AFM) techniques have recently been explored for several single cell manipulations. For example, the introduction of Bovine Serum Albumin molecules into cells by local electroporation [5] and the detection of -actin molecules (copy number 2000-3000) adsorbed on AFM probes inserted into living cells; although very interesting, these experiments were limited to high copy number detection since the AFM probe picks up all molecules that are non-specifically and randomly adsorbed to its surface [6]. We have developed a selective and ultra fast method to extract mRNA molecules from a single living cell down to very low copy numbers (10 molecules per cell). The technology builds around the Atomic Pressure Sagopilone Microscope (AFM) platform. We modified the structure of a commercially available AFM probe tip to accommodate the electric field gradients necessary for extraction of the mRNA molecules through dielectrophoresis. Target-specific short mRNA capture primers (20-30 base pairs long) were immobilized around the modified AFM probes using Biotin-Streptavidin chemistry [7]. Cells were either plated on regular glass cover slides, micropallet arrays (MPA) or deposited on cover slides after trypsinazation. Upon inserting the modified probe tip into a selected single living cell, the target mRNA was captured by generating a near-field dielectrophoretic pressure at the end of the tip, to attract and hybridize target mRNA onto the tip. Following hybridization, the tip is usually withdrawn from the cell, and the extracted mRNA Sagopilone is usually collected and analyzed using conventional RT-PCR. We performed computer simulations to determine the strength and distribution of the dielectrophoretic pressure on ssDNA molecules as shown inFigure 1(c)[7] ; note that molecules are attracted towards structurally and chemically modified area of the tip where capture primers are immobilized. == Determine 1. == (A) Schematic representation of the experimental set-up used in mRNA extraction (not to a scale). (B) Scanning Electron Microscope (SEM) image of the modified tip. Note that inner Si and outer Pt are seen in the picture. An electric field is usually applied between inner Si and out Pt to establish a dielectrophoretic pressure. Scale bar is usually 100 nm (C) FEM of mRNA capturing pressure. Direction of the pressure is usually shown in arrows. Scale bar is usually 100nm. (D) Indicates the cells immobilized around the MPA for mRNA extraction. This is combination of two images (bright field image and corresponding fluorescence image). Note that target cells (ratneuinjected cells) are fluorescently labeled. Scale bar indicates 10um. We modified Rabbit polyclonal to CD59 commercially available AFM tips (k ~ 1.5N/m, tip length ~ 10 um, end curvature ~20nm and 0.001 -cm from Veeco probes, model: 1930-00) in the following manner. A 20nm solid layer Sagopilone of SiO2 was grown in a conventional oxidation furnace (60 min at 900 C) to electrically insulate the entire probe tip surface including the AFM cantilever. 10nm of Ti and 10 nm of Pt were e-beam evaporated around the oxidized tips. The Pt coated tip end was then sectioned until it barely exposed the SiO2.Determine 1(b)shows the final version of a tip after processing..