Demonstration of Turbo SNP FISH.

<p>A. Demonstration of SNP FISH efficacy under Turbo FISH and conventional RNA FISH conditions in WM983b cells. We targeted BRAF mRNA with guide probes, and then used detection probes that targeted either the V600E mutation for which BRAF is heterozygous in this cell line (top panels) or a common region for which BRAF is homozygous in this cell line (bottom panels). Left panels show the signals from the guide probe (that labels the mRNA), the middle panel shows the detection probe that detects the wild-type sequence, and the right panel shows the detection probe that detects the mutant sequence. B. Quantification of RNA as being either mutant or wild type in this cell line. Each bar corresponds to data from a single cell.</p

Depiction of the RNA FISH scheme and demonstration of rapid hybridization.

<p>A. Schematic of the single molecule RNA FISH method, in which we use dozens of short fluorescently labeled oligonucleotides that all target the same RNA molecule. B. Image showing RNA FISH targeting mRNA from the <i>TBCB</i> gene under standard overnight hybridization conditions (formaldehyde fixation). Each spot is a single mRNA molecule. C. Image showing RNA FISH signals from an attempt at rapid hybridization (5 minutes) with a high concentration of probe but with formaldehyde fixation. D., E. Traditional overnight hybridization and Turbo RNA FISH hybridization using methanol-fixed cells. All images are maximum projections of a stack of optical sections encompassing the three-dimensional volume of the cell. DAPI (nuclear stain) is in purple.</p

Quantification of signal quality and comparison of different hybridization times and probe concentrations.

<p>A. Schematic depicting the manner in which we quantify signal quality via threshold sensitivity. B. Sensitivity of threshold measured in varying probe concentrations and hybridization times. The dotted line represents the sensitivity of a traditional overnight RNA FISH. Error bars reflect standard error of the mean. C. Spot counts for the same conditions as in B. Error bars reflect standard deviation. At 10 minutes and for all probe concentrations, the spot counts for Turbo FISH are statistically different from overnight FISH (4X: p = 9.87×10⁻⁶, 1X: p = 0.0136, 1/4X: p = 4.86×10⁻⁶, 1/16X: p = 1.75×10⁻¹¹; two-tailed t-test). For all conditions, we analyzed spot counts and calculated the sensitivity on 80–120 cells. Data shown represents one of two replicate experiments.</p

Demonstration of Turbo iceFISH.

<p>We performed Turbo FISH using iceFISH probes that targeted a total of 20 introns in genes on chromosome 19 (right panels), while simultaneously performing RNA FISH for TOP2A mRNA (left panels). We compared both Turbo FISH to conventional RNA FISH performed overnight (top vs. bottom panels). All images are maximum projections of a stack of optical sections encompassing the three-dimensional volume of the cell. DAPI (nuclear stain) is in blue.</p

Comparison of fixation conditions for both traditional overnight hybridizations and rapid hybridization.

<p>A. Comparison of number of spots detected and cumulative distribution functions for the <i>TBCB</i> gene with probes labeled with the Alexa 594 fluorophore. Error bars represent the standard error of the mean. No statistically significant differences exist between the overnight RNA FISH samples. Turbo RNA FISH for <i>TBCB</i> gene on formaldehyde-fixed cells is statistically different from Turbo RNA FISH on methanol- and ethanol-fixed cells (p = 3.82×10⁻⁶⁵ and p = 4.89×10⁻⁹⁶, respectively; two-tailed t-test). For all conditions, we analyzed spot counts on 100–150 cells. B. Comparison of number of spots detected and cumulative distribution functions for the <i>TOP2A</i> gene with probes labeled with the Cy3 fluorophore. Error bars represent the standard error of the mean. Overnight RNA FISH for <i>TOP2A</i> gene on formaldehyde-fixed cells is statistically different from overnight RNA FISH on ethanol-fixed cells (p = 0.0067; two tailed t-test). No other statistically significant differences exist between overnight RNA FISH samples. Turbo RNA FISH for <i>TOP2A</i> gene on formaldehyde-fixed cells is statistically different from Turbo RNA FISH on methanol- and ethanol-fixed cells (p = 9.57×10⁻²⁸ and p = 4.22×10⁻³⁰, respectively; two-tailed t-test). For all conditions, we analyzed spot counts on 100–150 cells. Data shown represents one of two replicate experiments.</p

Comparison of signal from Turbo RNA FISH (5 minutes; red) to conventional RNA FISH (blue).

<p>A. Comparison of RNA FISH signal sensitivity at a range of hybridization times. Error bars reflect standard error of the mean. At 5 minutes, we found a statistically significant difference in signal sensitivity between Turbo FISH and conventional FISH for <i>TBCB</i> gene and <i>TOP2A</i> gene (p = 4.75×10⁻¹¹ and p = 1.19×10⁻⁷⁴, respectively; two-tailed t-test). B. Comparison of RNA FISH spot count at a variety of hybridization times. Error bars reflect standard deviation. At 5 minutes, we found a statistically significant difference in RNA FISH spot count between the Turbo FISH and conventional FISH for <i>TBCB</i> gene and <i>TOP2A</i> gene (p = 1.69×10⁻⁶⁸ and p = 2.07×10⁻²⁰, respectively; two-tailed t-test). For all conditions, we analyzed spot counts and calculated sensitivity on 100–150 cells. Data shown represents one of two replicate experiments.</p