False negative rates in Drosophila cell-based RNAi screens: a case study

<p>Abstract</p> <p>Background</p> <p>High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.</p> <p>Results</p> <p>We performed a meta-analysis of several genome-wide, cell-based <it>Drosophila </it>RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.</p> <p>Conclusions</p> <p>RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.</p

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated

Strong field double ionization of atoms below the recollision threshold

Double ionization of Ar and Ne by 45 fs laser pulses (800 nm) has been explored at low intensities where ionization as a result of electron recollision is energetically not possible. We observe a strongly correlated back-toback emission of the electrons along the polarization direction in striking contrast to all previous data at higher intensities and most calculations

Strong-field double ionization of Ar below the recollision threshold

Nonsequential double ionization of Ar by 45 fs laser pulses (800 nm) at (4-7)×1013W/cm2 was explored in fully differential measurements. Well below the field-modified recollision threshold we enter the multiphoton regime. Strongly correlated back-to-back emission of the electrons along the polarization direction is observed to dominate in striking contrast to all previous data. No effect of Coulomb repulsion can be found, the predicted cutoff in the sum-energy spectra of two emitted electrons is confirmed, and the potential importance of multiple recollisions is discussed. © 2008 The American Physical Society

Multiphoton double ionization of Ar and Ne close to Threshold

In kinematically complete studies we explore double ionization (DI) of Ne and Ar in the threshold regime (I>3×1013W/cm2) for 800 nm, 45 fs pulses. The basic differences are found in the two-electron momentum distributions-"correlation" (CO) for Ne and " anticorrelation" (ACO) for Ar-that can be partially explained theoretically within a 3D classical model including tunneling. Transverse electron momentum spectra provide insight into "Coulomb focusing" and point to correlated nonclassical dynamics. Finally, DI threshold intensities, CO as well as ACO regimes are predicted for both targets. © 2010 The American Physical Society

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degefieration(1-5). PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed(6). Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families(7,8). Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16013.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes(10). Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6)