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

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

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

Activation curves of the mutant and wild type sodium channels at 25°C and 35°C.

<p>(A–D) Activation curves of wild type and I136V, I848T, V1316A mutant channels at 25°C (solid dots) and 35°C (open circles), respectively. (E) Wild type and V1316A mutant channels show a hyperpolarizing shift (WT: ∼4.2 mV; V1316A: ∼3.7 mV) in activation V_1/2 at 35°C compared with 25°C, while I136V is unchanged. I848T mutant channel shows a depolarizing shift (∼4 mV) at 35°C. All activation V_1/2 of mutant channels are more hyperpolarized than wild type at 25°C. (F) The slope of V1316A significantly reduces at 35°C as compared with 25°C. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. wild type; ^#<i>P</i><0.05, ^###<i>P</i><0.001 vs. 35°C; <i>t</i>-test; data shown as means ± SEM.</p

Inactivation recovery rate of wild type and mutant Na_v1.7 channels.

<p>(A) Recovery time constant τ is 10.7 ms for wild type channel (solid circles). (B) I136V mutant channel has significantly faster recovery from inactivation between inerstimulus interval of 4–12 ms comapred with wild type channel and has a recovery τ of 4.25 ms. (C) I848T mutant channel recovers from inactivation significantly faster than wild type (solid circles) between 4–8 ms of interstimulus interval with a recovery τ, 5.58 ms. (D) V1316A mutant channel has significantly faster recovery from inactivation between interstimulus interval of 4–12 ms and has a recovery τ of 5.29 ms. N numbers are annotated in parentheses; *<i>P</i><0.05, **<i>P</i><0.01 vs. wild type; <i>t</i>-test; data shown as means ± SEM.</p

Steady-state fast inactivation curves of wild type and the mutant channels at 25°C and 35°C.

<p>(A–D) Steady-state fast inactivation curves of wild type and I136V, I848T, V1316A mutant channels at 25°C (solid squares) and 35°C (open circles), respectively. (E) No significant difference in inactivation V_1/2 of wild type channel between 25°C and 35°C. At 35°C all mutant channels produce a significantly more depolarized inactivation V_1/2 compared with 25°C. (F) There is no significant difference in slopes. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. wild type; ^#<i>P</i><0.05, ^###<i>P</i><0.001 vs. 35°C; <i>t</i>-test; data shown as means ± SEM.</p

A Novel <em>SCN9A</em> Mutation Responsible for Primary Erythromelalgia and Is Resistant to the Treatment of Sodium Channel Blockers

<div><p>Primary erythromelalgia (PE) is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or exercise. Mutations in human <em>SCN9A</em> gene, encoding the α–subunit of the voltage-gated sodium channel, Na_v1.7, were found to be responsible for PE. Three missense mutations of <em>SCN9A</em> gene have recently been identified in Taiwanese patients including a familial (I136V) and two sporadic mutations (I848T, V1316A). V1316A is a novel mutation and has not been characterized yet. Topologically, I136V is located in DI/S1 segment and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the elelctrophysiological manifestations, the channel conductance with whole-cell patch clamp was recorded on the over-expressed Chinese hamster overy cells. As compared with wild type, the mutant channels showed a significant hyperpolarizing shift in voltage dependent activation and a depolarizing shift in steady-state fast inactivation. The recovery time from channel inactivation is faster in the mutant than in the wild type channels. Since warmth can trigger and exacerbate symptoms, we then examine the influence of tempearture on the sodium channel conduction. At 35°C, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift at activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. High temperature caused a significant depolarizing shift in steady-state fast inactivation in all three mutant channels. These findings may confer to the hyperexcitability of sensory neurons, especially at high temperature. In order to identifying an effective treatment, we tested the IC₅₀ values of selective sodium channel blockers, lidocaine and mexiletine. The IC₅₀ for mexiletine is lower for I848T mutant channel as compared to that of the wild type and other two mutants which is comparable to the clinical observations.</p> </div