Depression and family support in breast cancer patients

MTS, migration and invasion assays in DCIS.COM cells that were previously transduced with scrambled control (Control) or BCL9 KD shRNA. The control cells and BCL9 KD cells were re-transduced with empty vector (EV), BCL9 overexpression (BCL9-OE) and BCL9 KD. BCL9-OE was achieved by transduction using the PCDH-BCL9 (BCL9-OE) acquired from Dr. Carrasco [11]. A Western blot analysis was performed using anti-BCL9, anti-vimentin, anti-E-cadherin antibodies, and anti-β-actin as a loading control. B MTS assay on control cells transduced with EV (control + EV), or BCL9-OE (control + BCL9-OE), BCL9-KD transduced with EV (BCL9 KD + EV), and BCL9-KD transduced with BCL9-OE (BCL9 KD + BCL9-OE). Bar graphs represent mean absorbance at 490 nm normalized to control ± standard error of the mean (SEM) (n = 6). C, D Representative images of the migration and invasion assays. Bar graph represents percent area of cells migrated (left) and invaded (right) under the membrane after 24 h. Invasion and migration were determined by ImageJ analysis of microscopic images per sample, the data are mean values normalized to control ± SEM (n = 3). E TopFlash and FopFlash reporter activity in DCIS.COM transduced as above that were either treated with Wnt3A or control conditioned medium (CM). Data represent mean ± SEM (n = 3, letters indicate statistically significant difference). (PDF 964 kb

Digital PCR Modeling for Maximal Sensitivity, Dynamic Range and Measurement Precision

<div><p>The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (P_o) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λ_L) to one negative (λ_U) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.</p></div

Effect of False Reaction Calls on Precision and Dynamic Range.

<p>False positives effect precision curves differently from false negatives. False positive rates are represented in the plot via shapes (triangles = 0.0%, circles = 0.1%). False negative rates are represented by colors (red = 0.0%, green = 0.1%, blue = 0.2%). Note that false positives strongly impact precision at lower concentrations (where there are very few positives) and false negatives strongly impact precision at higher concentrations (where there are very few negatives)</p

Lower Limit of Detection dependence on False Positive Rate.

<p>Lower limit of detection is influenced most heavily by false positives. This dependence is due to limited effect false negatives have in counter balancing false positives due to the limited number of positives at lower concentrations. The plot demonstrates the effects of false reaction call rates on the lower limit of detection for 20,000 partitions at 20% precision. Using a baseline of 0% false positives and 0% false negatives, the lower limit of detection is measured at 0.006 copies/partition. Raising the false positive rate to 1% results in the raising of the lower limit of detection to 0.065 copies/partition, over an order of magnitude elevation.</p

Sample, Assay, Reaction Mix and Thermal Protocol Information.

<p>Sample, Assay, Reaction Mix and Thermal Protocol Information.</p

Gains in Dynamic Range via Increasing Number of Dilutions—Constant Available Partitioning.

<p>Fig. shows the benefits to dynamic range spanned by increasing the number of dilution steps across available partitioning. X-axis depicts an increasing number of dilution steps spread across a constant (20,160) number of available partitions. Subsets are assumed to be equally sized. As partitions per subset decreases with increasing number of subsets, boxed dilution values represent optimized serial dilutions (# subsets—1 steps) ensuring maximum Dynamic Range gain while maintaining overlapping regions of detection. Benefits to Dynamic Range are slight at high levels of precision (e.g., 5%) and are even seen to occasionally decrease as subsets increase (e.g., 5% precision between 5 and 6 subsets). However, substantial improvement is also seen as precision requirements are relaxed.</p

Digital PCR Fold Change between Adjacent Samples.

<p>Fig. shows the fold change between digital PCR computed quantities for all adjacent samples (e.g., between Sample A and Sample B). The values are seen to compare very well with the expected 6.8 fold difference between samples.</p

Trials with Two Dilution Factors to Select Optimal One For Use with Paired Dilution Strategy.

<p>Fig. shows that the continuous detection criterion fails at the application of the 1:1000 dilution factor. The 1:200 dilution factor meets the continuous detection criterion at a 10% precision requirement.</p

Workflow to Select Dilution Factor For Paired Chip Strategy for Maximizing Dynamic Range.

<p>Fig. shows how to select the dilution factor for the second chip in a pairing strategy to maximize the dynamic range.</p

Quantitative PCR C_Q Results of Original Dilution Series.

<p>Fig. shows computed CQ results for the 5 Unknown samples using the quantitative PCR. The CQ values correspond to a dynamic range of 3.393 logs between Samples A and Sample E and support the digital PCR results.</p