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Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.qPCR Technique: The Perception and Reality qPCR (see Glossary) is generally viewed by researchers as a powerful technique that can provide precise and quantitative data reflecting the biology of the tested experimental parameters. However, without following strict guidelines, validation and data analysis procedures, the results can be far from valid [1,2]. Unfortunately, the adoption and transfer of inadequate and varied protocols between individual laboratory members and laboratories throughout the scientific community have led to frustration in reproducing data [3][4][5]. This has driven the production of the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines and related methodology articles to help the scientific community in augmenting experimental rigor and uniformity to produce more reliable and consistent data [6][7][8]. Nevertheless, there remain concerns regarding the quality of qPCR results in the published literature [1,2].When designing experiments for qPCR, all protocols, such as sample handling, harvesting, nucleic acid extraction, reverse transcription, and qPCR should be described and vetted in detail. Mistakes or assumptions can be made in the planning process, resulting in a flawed experimental design with results and conclusions based on artefacts of pre and/or post sample handling procedures as opposed to the true effect of the tested experimental parameters [7]. Poorly optimized reactions can result in data that are consequent to a combination of sample contaminants and/or poor annealing temperature, leading to misinterpreted results and conclusions that are difficult or even impossible to reproduce [9,10].Despite the MIQE guidelines and other methodology articles, the variability and reproducibility pitfalls associated with qPCR remain elusive for many laboratories [7,11]. This review article describes the major sources of error associated with a qPCR experiment and strategies for their Highlights qPCR is more complex than perceived by many scientists.

Current methods of assessing psychopathology depend almost entirely on verbal report (clinical interview or questionnaire) of patients, their family, or caregivers. They lack systematic and efficient ways of incorporating behavioral observations that are strong indicators of psychological disorder, much of which may occur outside the awareness of either individual. We compared clinical diagnosis of major depression with automatically measured facial actions and vocal prosody in patients undergoing treatment for depression. Manual FACS coding, active appearance modeling (AAM) and pitch extraction were used to measure facial and vocal expression. Classifiers using leave-one-out validation were SVM for FACS and for AAM and logistic regression for voice. Both face and voice demonstrated moderate concurrent validity with depression. Accuracy in detecting depression was 88% for manual FACS and 79% for AAM. Accuracy for vocal prosody was 79%. These findings suggest the feasibility of automatic detection of depression, raise new issues in automated facial image analysis and machine learning, and have exciting implications for clinical theory and practice..

Key Points After being killed by artesunate, malaria parasites are expelled from red cells and then these pitted red cells reenter the circulation. When many pitted red cells are produced during therapy, their delayed clearance a few weeks later triggers hemolytic episodes.

IntroductionDuring their 120-day life span, human RBCs repeatedly traverse capillaries of the vascular bed and interendothelial slits of the venous sinus of spleen red pulp, both of which are narrower than their smallest dimension. 1 This necessitates maintenance of the ability of RBCs to undergo repeated, extensive, and reversible deformations. Repeated major membrane deformations induce ion and water permeability changes in the RBCs. [2][3][4][5] The biconcave discoid shape endows the human RBC with an advantageous surface area-to-volume (S/V) ratio, allowing the cell to undergo marked deformations while maintaining a constant surface area. [6][7][8] A reduced RBC S/V ratio has long been recognized to contribute to pathogenesis of several RBC disorders, 9-11 including hereditary spherocytosis (HS), the most common cause of inherited chronic hemolytic anemia in Northern Europe and North America, with an estimated incidence of 1 in 2000. 11 The clinical presentation of HS can range from mild to severe hemolytic anemia. 12,13 The molecular basis of HS is heterogeneous, the common denominator being the loss of HS RBC membrane surface area due to specific molecular defects in several membrane proteins (␣ or ␤ spectrin, ankyrin, protein 4.2, and protein band 3), which result in the loss of cohesion between the lipid bilayer and the membrane skeleton. 10,11,14,15 The loss of membrane surface area results in the transformation of the biconcave discoid shape, first to a stomatocyte and finally to a spherocyte, with a progressive reduction in cellular deformability.Although a major role for the spleen in pathogenesis of HS is well established, 16 there are currently no data on the quantitative relationship between the extent of surface area loss and the extent of splenic entrapment. The only indirect evidence comes from early studies documenting reduced survival of Cr 51 -labeled spherocytes infused into healthy recipients, whereas the survival of normal RBCs in HS subjects was normal. [17][18][19] This implied that the reduced life span and splenic entrapment was an intrinsic feature of HS RBCs. Unfortunately, because neither the surface area nor the S/V ratio of infused spherocytes was measured in these studies, the extent of membrane surface area loss (or reduced S/V ratio) that leads to splenic retention of altered RBCs remains undefined. As a result, there is no predictive biologic parameter with which to estimate the risk of splenic entrapment of spherocytic cells and the ensuing anemia. Previously, we addressed this important issue using the isolated human spleen system 20 perfused with human RBCs with defined extents of surface area loss.RBCs with defined loss in membrane surface area can be generated experimentally by treatment with lysophosphatidylcholine (LPC). 6,7 By initially accumulating exclusively in the external leaflet of the RBC lipid bilayer, LPC induces in a dose-dependent manner echinocytosis There is an Inside Blood commentary on this article in this issue.The online version of this article contai...