Incorporating Primer Amplification Efficiencies in Quantitative Reverse Transcription Polymerase Chain Reaction Experiments; Considerations for Differential Gene Expression Analyses in Zebrafish.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is commonly used to measure the mRNA expression of target genes in zebrafish. Gene expression values from RT-qPCR are typically reported as relative fold-changes, and relative quantification of RT-qPCR data incorporates primer amplification efficiency values for each target gene. We describe the influence of the primer amplification efficiency analysis method on RT-qPCR gene expression fold-change calculations. This report describes (1) a sample analysis demonstrating incorporation of primer amplification efficiency into RT-qPCR analysis for comparing gene expression of a gene of interest between two groups when normalized to multiple reference genes, (2) the influence of differences in primer amplification efficiencies between measured genes on gene expression differences calculated from theoretical delta-Cq (dCq) values, and (3) an empirical comparison of the influence of three methods of defining primer amplification efficiency in gene expression analyses (delta-delta-Cq [ddCq], standard curve, LinRegPCR) using mRNA measurements of a set of genes in zebrafish embryonic development. Given the need to account for the influence of primer amplification efficiency along with the simplicity of using software programs (LinRegPCR) to measure primer amplification efficiency from RT-qPCR data, we encourage using empirical measurements of primer amplification efficiency for RT-qPCR analysis of differential gene expression in zebrafish.

Randomized controlled trial of mycophenolate mofetil in children, adolescents, and adults with IgA nephropathy.

BACKGROUND: Previous randomized controlled trials evaluating the efficacy of mycophenolate mofetil (MMF) in patients with immunoglobulin A nephropathy (IgAN) have produced varying results. STUDY DESIGN: Double-blind placebo-controlled randomized controlled trial. SETTING & PARTICIPANTS: 52 children, adolescents, and adults with biopsy-proven IgAN in 30 centers in the United States and Canada. Entry criteria: age older than 7 to younger than 70 years; urine protein-creatinine ratio (UPCR), ≥0.6g/g (males) or ≥0.8g/g (females); and estimated glomerular filtration rate ≥ 50mL/min/1.73m(2) (≥40mL/min/1.73m(2) if receiving angiotensin-converting enzyme inhibitor). Mean age, 32±12 (SD) years; 62% men; and 73% white. INTERVENTION: Lisinopril (or losartan) plus a highly purified omega-3 fatty acid (Omacor [Pronova Biocare]) was given to 94 patients for 3 months; 52 of the patients with persistent UPCR≥0.6g/g (males) and ≥0.8g/g (females) were randomly assigned to MMF or placebo (target dose, 25-36mg/kg/d) in addition to lisinopril/losartan plus Omacor. OUTCOMES: Change in UPCR after 6 and 12 months treatment with MMF/placebo and 12 months after the end of treatment. MEASUREMENTS: UPCR measured on 24-hour urine samples. Glomerular filtration rate estimated with the Schwartz (age < 18 years) or Cockcroft-Gault (age ≥ 18 years) formula. RESULTS: 44 patients completed 6 months of treatment with MMF (n=22) or placebo (n=22). The trial was terminated early at the recommendation of the Data Monitoring Committee because of the lack of benefit. No patient achieved a complete remission (UPCR<0.2g/g). Mean UPCRs at randomization and after 6 months were 1.45 (95% CI, 1.16-1.75) and 1.40 (95% CI, 1.09-1.70) for MMF and 1.41 (95% CI, 1.17-1.65) and 1.58 (95% CI, 1.13-2.04) for placebo, respectively. The mean difference in UPCR change between these groups (MMF minus placebo) was -0.22 (95% CI, -0.75 to 0.31; P=0.4). Adverse events were rare apart from nausea (MMF, 8.7%; placebo, 3.7%); one of these MMF patients withdrew. LIMITATIONS: Low patient enrollment and short follow-up. CONCLUSIONS: MMF did not reduce proteinuria significantly in patients with IgAN who had persistent proteinuria after lisinopril/losartan plus Omacor.

Smartphone-Based Android app for Determining UVA Aerosol Optical Depth and Direct Solar Irradiances.

This research describes the development and evaluation of the accuracy and precision of an Android app specifically designed, written and installed on a smartphone for detecting and quantifying incident solar UVA radiation and subsequently, aerosol optical depth at 340 and 380 nm. Earlier studies demonstrated that a smartphone image sensor can detect UVA radiation and the responsivity can be calibrated to measured direct solar irradiance. This current research provides the data collection, calibration, processing, calculations and display all on a smartphone. A very strong coefficient of determination of 0.98 was achieved when the digital response was recalibrated and compared to the Microtops sun photometer direct UVA irradiance observations. The mean percentage discrepancy for derived direct solar irradiance was only 4% and 6% for observations at 380 and 340 nm, respectively, lessening with decreasing solar zenith angle. An 8% mean percent difference discrepancy was observed when comparing aerosol optical depth, also decreasing as solar zenith angle decreases. The results indicate that a specifically designed Android app linking and using a smartphone image sensor, calendar and clock, with additional external narrow bandpass and neutral density filters can be used as a field sensor to evaluate both direct solar UVA irradiance and low aerosol optical depths for areas with low aerosol loads.

Evaluating UVA aerosol optical depth using a smartphone camera.

This research evaluates a smartphone complementary metal oxide semiconductor (CMOS) image sensor's ability to detect and quantify incident solar UVA radiation and subsequently, aerosol optical depth at 340 and 380 nm. Earlier studies revealed that the consumer grade CMOS sensor has inherent UVA sensitivities, despite attenuating effects of the lens. Narrow bandpass and neutral density filters were used to protect the image sensor and to not allow saturation of the solar images produced. Observations were made on clear days, free from clouds. The results of this research demonstrate that there is a definable response to changing solar irradiance and aerosol optical depth can be measured within 5% and 10% error margins at 380 and 340 nm respectively. The greater relative error occurs at lower wavelengths (340 nm) due to increased atmospheric scattering effects, particularly at higher air masses and due to lower signal to noise ratio in the image sensor. The relative error for solar irradiance was under 1% for observations made at 380 nm. The results indicate that the smartphone image sensor, with additional external narrow bandpass and neutral density filters can be used as a field sensor to evaluate solar UVA irradiance and aerosol optical depth.

Characterization of a smartphone camera's response to ultraviolet A radiation.

As part of a wider study into the use of smartphones as solar ultraviolet radiation monitors, this article characterizes the ultraviolet A (UVA; 320-400 nm) response of a consumer complementary metal oxide semiconductor (CMOS)-based smartphone image sensor in a controlled laboratory environment. The CMOS image sensor in the camera possesses inherent sensitivity to UVA, and despite the attenuation due to the lens and neutral density and wavelength-specific bandpass filters, the measured relative UVA irradiances relative to the incident irradiances range from 0.0065% at 380 nm to 0.0051% at 340 nm. In addition, the sensor demonstrates a predictable response to low-intensity discrete UVA stimuli that can be modelled using the ratio of recorded digital values to the incident UVA irradiance for a given automatic exposure time, and resulting in measurement errors that are typically less than 5%. Our results support the idea that smartphones can be used for scientific monitoring of UVA radiation.

A high-affinity reversible protein stain for Western blots.

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.