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

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

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

Transcriptional Bursting Explains the Noise–Versus–Mean Relationship in mRNA and Protein Levels

<div><p>Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.</p></div

Protein and mRNA noise are inversely correlated with abundance.

<p>(<b>A</b>) Re-plotting of [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158298#pone.0158298.ref005" target="_blank">5</a>] GFP protein measurements for 30 HIV LTR-GFP isoclonal cell populations each with a distinct genomic integration site. Each point represents ~3,000 clonal cells (extrinsic noise filtered out by sub-gating of 50,000) and clones fall along distinct hyperbolic manifolds of transcriptional burst that are analytical solutions to the two-state model where Burst Size = (CV² × ) / 5,000–1 as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158298#pone.0158298.ref005" target="_blank">5</a>]. Grey lines represent burst sizes from 0–12. Color lines are highlighted burst sizes. (<b>B</b>) 30 different LTR-d2GFP (2-hr half-life GFP) clonal populations before TNF-α (black) and after 18-hr TNF-α (red) exposure, reproduced from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158298#pone.0158298.ref007" target="_blank">7</a>] where extrinsic noise was filtered out as in A. As predicted from the two-state model, noise is constrained between hyperbolic manifolds of constant burst size (gray). Black lines represent min and max burst size lines fit to dimmest and brightest clones, respectively, before TNF-α exposure. Representative individual clones with a yellow border and labeled as I, II, and III. (<b>C</b>) Re-plotting of Dey et al. (2015) smFISH RNA measurements for 23 LTR-GFP isoclones (Burst Size = (CV² × ) showing that clones fall along distinct burst model lines. (<b>D</b>) New smFISH analysis of LTR-d₂GFP mRNA for eight different clones (a subset of isoclones originally reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158298#pone.0158298.ref007" target="_blank">7</a>]) before TNF-α (black) and after 18-hr TNF-α (red) exposure. Yellow border clones I, II, and III are the same clones as in panel B and black lines calculated as in panel B. A summary table detailing the origin of the data in each panel appears in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158298#pone.0158298.s001" target="_blank">S1 File</a>.</p