Model based analysis of real-time PCR data from DNA binding dye protocols

BACKGROUND: Reverse transcription followed by real-time PCR is widely used for quantification of specific mRNA, and with the use of double-stranded DNA binding dyes it is becoming a standard for microarray data validation. Despite the kinetic information generated by real-time PCR, most popular analysis methods assume constant amplification efficiency among samples, introducing strong biases when amplification efficiencies are not the same. RESULTS: We present here a new mathematical model based on the classic exponential description of the PCR, but modeling amplification efficiency as a sigmoidal function of the product yield. The model was validated with experimental results and used for the development of a new method for real-time PCR data analysis. This model based method for real-time PCR data analysis showed the best accuracy and precision compared with previous methods when used for quantification of in-silico generated and experimental real-time PCR results. Moreover, the method is suitable for the analyses of samples with similar or dissimilar amplification efficiency. CONCLUSION: The presented method showed the best accuracy and precision. Moreover, it does not depend on calibration curves, making it ideal for fully automated high-throughput applications

Intrinsically Photosensitive Retinal Ganglion Cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light in the absence of all rod and cone photoreceptor input. The existence of these ganglion cell photoreceptors, although predicted from observations scattered over many decades, was not established until it was shown that a novel photopigment, melanopsin, was expressed in retinal ganglion cells of rodents and primates. Phototransduction in mammalian ipRGCs more closely resembles that of invertebrate than vertebrate photoreceptors and appears to be mediated by transient receptor potential channels. In the retina, ipRGCs provide excitatory drive to dopaminergic amacrine cells and ipRGCs are coupled to GABAergic amacrine cells via gap junctions. Several subtypes of ipRGC have been identified in rodents based on their morphology, physiology and expression of molecular markers. ipRGCs convey irradiance information centrally via the optic nerve to influence several functions including photoentrainment of the biological clock located in the hypothalamus, the pupillary light reflex, sleep and perhaps some aspects of vision. In addition, ipRGCs may also contribute irradiance signals that interface directly with the autonomic nervous system to regulate rhythmic gene activity in major organs of the body. Here we review the early work that provided the motivation for searching for a new mammalian photoreceptor, the ground-breaking discoveries, current progress that continues to reveal the unusual properties of these neuron photoreceptors, and directions for future investigation

Population-based screening for cancer: hope and hype

Several important lessons have been learnt from our experiences in screening for various cancers. Screening programmes for cervical and colorectal cancers have had the greatest success, probably because these cancers are relatively homogenous, slow-growing, and have identifiable precursors that can be detected and removed; however, identifying the true obligate precursors of invasive disease remains a challenge. With regard to screening for breast cancer and for prostate cancer, which focus on early detection of invasive cancer, preferential detection of slower-growing, localized cancers has occurred, which has led to concerns about overdiagnosis and overtreatment; programmes for early detection of invasive lung cancers are emerging, and have faced similar challenges. A crucial consideration in screening for breast, prostate, and lung cancers is their remarkable phenotypic heterogeneity, ranging from indolent to highly aggressive. Efforts have been made to address the limitations of cancer-screening programmes, providing an opportunity for cross-disciplinary learning and further advancement of the science. Current innovations are aimed at identifying the individuals who are most likely to benefit from screening, increasing the yield of consequential cancers on screening and biopsy, and using molecular tests to improve our understanding of disease biology and to tailor treatment. We discuss each of these concepts and outline a dynamic framework for continuous improvements in the field of cancer screening