Indophenol Blue Colorimetric Method to Determine Grain Protein Content of Cereal Plants.

Here, we propose a method to convert the organic nitrogen in maize kernels into ammonia in solution and then chlorinate it to prepare monochloride salts, which can form an oxidatively coupled blue-green mixture with sodium salicylate and sodium dichloroisocyanurate. The concentration of ammonium ions in the blue-green mixture can then be determined in the solution, and finally the protein content in maize kernels can be calculated from the nitrogen content.

Placental weight percentile curves in a Danish population.

INTRODUCTION: The role of the placenta is to transport oxygen and nutrients to the fetus, and a well-functioning placenta is vital to fetal health. Our aim was to develop placental weight percentile curves adjusted by gestational age, and stratified by major maternal comorbidities. MATERIAL AND METHODS: The study was a population study in a Danish cohort. Data was drawn from The Medical Birth Register and the National Patient Register. We included singleton births with a gestational age of 22 + 0 to 42 + 6 weeks. We excluded multiple pregnancies, stillbirths and retained placentas. A total of 611 418 placentas were included. Percentile line graphs were created in groups of all placentas, hypertensive disorders and diabetic disorders. RESULTS: Tables and figures are presented for placental weight percentile curves according to gestational age for all placentas, hypertensive disorders and diabetic disorders, respectively. Placental weight was generally higher in the diabetic placentas, and lower in the hypertensive placentas. CONCLUSIONS: These percentile curves may serve as a reference for other populations, and may be useful for other studies investigating the role of the placenta in relation to pregnancy outcomes, and health in later life.

Quantitative PCR of Alu Repeats Using PowerUp™ SYBR® Green Master Mix.

Quantitative PCR is one of the fundamental steps performed when processing routine casework in a forensic laboratory. Quantitative PCR of Alu repeats using a SYBR® Green master mix can produce calculated estimates of how much DNA was extracted from a sample. This process offers more efficiency, human specificity, and can be performed faster than other outdated quantification methods, such as slot blot or yield gel. A qPCR master mix is prepared and consists of Alu-F primers, Alu-R primers, water, and SYBR® Green master mix. The Alu-F and Alu-R primers target Alu sequences that are present hundreds of thousands of times throughout the human genome and are effective markers for human DNA quantification. During qPCR, the 7500 system facilitates the amplification of target Alu repeats. The SYBR® Green I fluorescent dye intercalates between the amplified dsDNA targets. During each amplification cycle, the 7500 system agitates the SYBR® Green I dye, resulting in a fluorescence signal that is recorded when it passes a specified Ct value. After qPCR amplification is complete, a standard curve is created and used to determine how much DNA a sample contains. This chapter provides instructions on how to accurately prepare a 96-well plate for qPCR, use the 7500 system and associated software to set up the qPCR amplification, and interpret the corresponding results produced.

Introduction to the Use of Linear and Nonlinear Regression Analysis in Quantitative Biological Assays.

Biological assays are essential tools in biomedical and pharmaceutical research. In simplest terms, such an assay is an analytical method used to measure or predict a response in a biological system in the presence of a given stimulus (e.g., drug). The inherent complexity involved in evaluating a biological system requires the use of rigorous and appropriate tools for data analysis. Linear and nonlinear regression models represent critically important statistical analyses used to define the relationships between variables of interest in biological systems. Recent challenges relating to the reproducibility of published data suggest the absence of standardized and routine use of statistics to support experimental results across a wide range of scientific disciplines. The current situation warrants an introductory review of basic regression concepts using current, practical examples, along with references to in-depth resources. The goal is to provide the necessary information to help standardize the analysis of biological assays in academic research and drug discovery and development, elevating their utility and increasing data transparency and reproducibility. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Sources of variance in the results of a commercial bovine immunoglobulin G radial immunodiffusion assay.

Radial immunodiffusion (RID) is used to quantify IgG concentration in neonatal beef or dairy calf serum; variability has been noted that may affect the precision and accuracy of assay results. We determined the source, range, and homogeneity of variance in the results of a commercial bovine IgG RID assay (Triple J Farm). To estimate the variance in the precipitin ring diameter, we used 6 sera, measured 28 times across 8 plates and 4 lots, and 3 standards with known IgG concentrations, measured 75 times across 69 plates and 5 lots. The source of diameter variance was determined using variance partition coefficients for lot, plate, and repetition. We used 11 different methods to generate standard curves to convert RID precipitin ring diameters to IgG concentrations. The Levene test of homogeneity of variance (α = 0.1) was used to evaluate the equality of variance between the standards or serum precipitin ring diameters and calculated IgG concentrations. Lot and plate contributed minimally to the diameter variance. Precipitin ring diameters had equal variance. Calculated IgG concentrations for serum not requiring dilution had equal variance. A linear equation from aggregated standards, performed within the same day, had greater accuracy for the calculated IgG concentrations of the standards compared to other equation methods. Regardless of standard curve methodology or IgG concentration, variability inherent to the assay limits its clinical usefulness.

Abductive statistical methods improve the results of calibration curve bioassays: An example of determining zinc bioavailability in broiler chickens.

In this paper, we discuss the theory behind calibration curve experiments and their application to a zinc (Zn) bioavailability study with broiler chickens. Seven replicates of 16 male commercial broiler chicks were fed starter diets for 14 days. Six diets had different levels of a potential Zn source and one was a positive control with standard industry levels of Zn for comparison. Four commonly used methods of calculating bioavailability means and confidence intervals (CI) from a calibration curve (standard curve) experiment to estimate the bioavailability of a new zinc source in broiler chickens were compared. The methods compared were the following: 1) the Counter-Intuitive Method uses a multiple-range test to compare unknown test and standard samples; 2) the Intuitive Method uses standard linear regression and inverts the equation to predict Zn bioavailability for each replicate of test samples; 3) the Abductive Method uses Graybill's Equation, based on theory and observation, to estimate CI's; and 4) the Sophistic Method uses reverse regression, and calculates Zn bioavailability values directly from the equation. The Counter-Intuitive Method only gives information about which standards the test samples are, or are not, significantly different from respectively (average available Zn not predicted). The Intuitive Method ignores error about the standard curve and theoretically cannot estimate the CI directly ( X ¯ ± SEM  = 107.5 ± 15.8 mg Zn/kg). The Sophistic Method underestimates and overestimates the test sample mean values above and below the mean of the standards, respectively ( X ¯  = 96.6 mg Zn/kg). The Abductive Method has an advantage over the other methods: The mean prediction estimation is consistent with theory (107.5 ± 6.1 mg Zn/kg; X ¯ ± SEM ). When test or "unknown" samples are near the mean of the standard samples, the CI is smaller than when near the extremes of the calibration curve. When calibration curve error is small (R 2 > approximately 0.95), there is little advantage to using the Abductive Method, but when calibration curve error is larger, as in many bioassays with growing animals, the Abductive Method improves the accuracy of the CI calculations. The Abductive Method was used to demonstrate the influence of the number of replicate samples on experimental power and cost.

Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues.

Reverse-phase high-performance liquid chromatography (HPLC) is a preferred method used to identify and quantify carotenoids. Here, we describe a straightforward, reliable, and cost-effective protocol to purify and develop individual carotenoid standards for absolute quantification of carotenoids, including selected cis-trans (geometric) isomers. Analytical techniques to extract, purify and collect individual carotenoids using an HPLC system equipped with a Diode Array Detector (DAD) and fraction collector are described. Carotenoids were separated and identified by their characteristic ultraviolet-visible (UV-Vis) absorption spectra and individually isolated based on their retention times using a C30 column. This chapter outlines how to prepare standard calibration curves using known quantities of purified and/or commercially available carotenoids. A series of molar extinction and slope coefficients for phytoene, phytofluene, ζ-carotene, neurosporene, pro-lycopene, all trans-lycopene, lutein, ß-carotene, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, capsanthin, capsorubin and ß-cryptoxanthin are defined to enable absolute quantification of their abundance in plant, animal, and bacterial tissues. Different approaches for reporting carotenoid abundance by absolute concentration, relative composition, and/or using ratios of different pigments are provided as a convenient resource for carotenoid researchers.

An analysis of the standard curve parameters of cytomegalovirus, BK virus and hepatitis B virus quantitative polymerase chain reaction from a clinical virology laboratory in eastern India.

PURPOSE: Quantitative real-time polymerase chain reactions (qPCRs) are important for accurate detection of nucleic acid target including that for viral load determination. Assessment of the quality of a PCR run is essential for quality control, diagnostics and research. In order to reduce subjectivity qPCR standard curves are accompanied with parametric values for slope, Y- intercept, correlation coefficient (R2) and PCR efficiency. In this study the performance of three qPCR assays-cytomegalovirus, hepatitis B virus and BK virus-with respect to standard curve parameters-slope, Y intercept, R2 and efficiency were examined. METHODS: Using ideal values (Slope (minus 3.32); Y intercept â€‹= â€‹the number of PCR cycles; R2 â€‹= â€‹1 and efficiency â€‹= â€‹100%) we estimated the intra-assay variability (range) and deviation from ideal parameters (Δ). We also calculated the standard deviation (SD) and coefficient of variation (CV) for each of these parameters. We have evaluated the quality of each of the three viral load assays (CMV, HBV, BKV) using these statistical approach. RESULTS: We found lab developed tests (CMV) to have least deviation from ideal Y intercept (limit of detection); however, commercial kit based assays had better linearity (scatter plot correlation between amplification factor and PCR efficiency). Using a scatter plot for the three assays we found the correlation with calculated amplification factor and PCR efficiency was most linear in case of BKV (0.9974), closely followed by the HBV assay (R2 â€‹= â€‹0.9968). Although the CMV quantitative standards were least linear (0.868), the CV (coefficient of variation) was also the least in case of the CMV assay. CONCLUSION: The study highlights an objective way of assessing qPCR assay quality and demonstrates a method to compare assays, validate tests and perform quality control.

Evaluation on measurement uncertainty of correction factors of fluconazole impurities determined by HPLC standard curve method / 中国药科大学学报

@#In this paper, the uncertainties of correction factors of fluconazole impurities determined by HPLC standard curve method were evaluated, and the main common factors affecting the accuracy of standard curve method were found, so as to improve the accuracy of the method.In this study, the corresponding fitting lines of fluconazole and its impurities A, B, C, D, F and I were established respectively, and the ratio of the slope of fitting lines of each impurity and its corresponding principal component was calculated as the correction factor of the impurity.Then on the basis of GUM method, the uncertainty of each impurity correction factor determined by standard curve method was evaluated according to the established uncertainty evaluation scheme of correction factor determination process.The correction factor and uncertainty of fluconazole impurities A, B, C, D, F and I were 1.068 ± 0.046, 0.102 ± 0.005, 0.0582 ± 0.0031, 1.382 ± 0.121, 0.802 ± 0.067 and 1.383 ± 0.119, respectively, and the coverage factor k was 2.Finally, the contribution rate of each uncertainty component was calculated.In the relative combined standard uncertainties urel(f) of fluconazole impurities A, B, C, D, F and I correction factors, the sum of contribution rate of slope uncertainty urel(K) of the linear equation of principal component and its impurity is more than 85%; in the slope uncertainties urel(K) of linear equation, the contribution rates of uncertainties of solution concentration in 8 of 12 data groups are more than 80%, and the contribution rates of uncertainties introduced by reference substance content in solution concentration are about 80%.It can be seen that the preparation of linear solution concentration is the most influential factor in the determination of impurity correction factor by standard curve method, followed by the linear fitting process.In the preparation process of linear solution concentration, the purity of reference substance is the most influential factor, followed by weighing and pipetting times.The conclusion can help the experimenters to better formulate experimental plans and ensure the accuracy of the results when doing similar work.

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance.

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.

Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics.

The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.

Extraction and Quantification of Sphingolipids from Hemiptera Insects by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules of sphingolipids, have been depicted in cell growth arrest, cell differentiation, and apoptosis. With the development of lipidomics, the identification of ceramides has been analyzed in many species, mostly in model insects. However, there is still a lack of research in non-model organisms. Here we describe a relatively simple and sensitive method for the extraction, identification, and quantification of ceramides in Hemiptera Insects (brown planthooper), followed by Ultra-Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). C18 is used as the separation column for quantitative detection and analysis on the triple quadruple liquid mass spectrometer. In this protocol, the standard curve method is adopted to confirm the more accurate quantification of ceramides based on the optional detection conditions.

Quantification of Ammonia Oxidizing Bacterial Abundances in Environmental Samples by Quantitative-PCR.

Quantitative-PCR (qPCR) enables the quantification of specific DNA targets, such as functional or phylogenetic marker genes associated with environmental samples. During each qPCR cycle, the number of copies of a gene (or region) of interest in DNA samples is determined in real time using a fluorescence-based label and compared to a standard serial dilution. Here, we describe a qPCR method to quantify the ammonia oxidizing bacteria involved in the first step of nitrification, using the amoA gene as a proxy of their abundance. The preparation of the standards from environmental samples and qPCR is presented in detail for specifically quantifying microbial abundance in environmental samples such as soil.

Simplified Analysis of Measurement Data from A Rapid E. coli qPCR Method (EPA Draft Method C) Using A Standardized Excel Workbook.

Draft method C is a standardized method for quantifying E. coli densities in recreational waters using quantitative polymerase chain reaction (qPCR). The method includes a Microsoft Excel workbook that automatically screens for poor-quality data using a set of previously proposed acceptance criteria, generates weighted linear regression (WLR) composite standard curves, and calculates E. coli target gene copies in test samples. We compared standard curve parameter values and test sample results calculated with the WLR model to those from a Bayesian master standard curve (MSC) model using data from a previous multi-lab study. The two models' mean intercept and slope estimates from twenty labs' standard curves were within each other's 95% credible or confidence intervals for all labs. E. coli gene copy estimates of six water samples analyzed by eight labs were highly overlapping among labs when quantified with the WLR and MSC models. Finally, we compared multiple labs' 2016-2018 composite curves, comprised of data from individual curves where acceptance criteria were not used, to their corresponding composite curves with passing acceptance criteria. Composite curves developed from passing individual curves had intercept and slope 95% confidence intervals that were often narrower than without screening and an analysis of covariance test was passed more often. The Excel workbook WLR calculation and acceptance criteria will help laboratories implement draft method C for recreational water analysis in an efficient, cost-effective, and reliable manner.

Use of synthetic oligonucleotides for determination of HTLV-1 proviral load by real-time PCR: a helpful alternative approach in the clinical management.

AIMS: To evaluate the potential use of synthetic oligonucleotides as a standard curve for proviral load (PVL) of human T-cell leukaemia virus type 1 (HTLV-1) quantification in peripheral blood mononuclear cells (PBMC) of HTLV-1-infected individuals by quantitative real-time polymerase chain reaction (qPCR) analysis. METHODS AND RESULTS: Synthetic oligonucleotides based on HTLV-1 genome were customized to use as a standard curve. Twelve anti-HTLV-1-positive samples with known HTLV-1 PVL, previously quantified by qPCR assay using TARL-2 cells as a conventional standard curve, were submitted to the new protocol. The proviral quantification levels had a high concordance with qPCR results using a conventional standard curve. The results demonstrate that the conventional standard curve can be replaced by a synthetic standard curve due to its ability to quantification based on the linearity and qPCR efficiency and similar results with a validated qPCR assay using a conventional standard curve. CONCLUSIONS: Synthetic oligonucleotides standard curves could be a very useful tool on HTLV-1 diagnosis and absolute HTLV-1 PVL quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: HTLV-1 PVL determination using synthetic oligonucleotides standard curve by qPCR could be a helpful alternative for the laboratories that monitor infected patients as an important prognostic factor in HTLV-1-associated diseases progression. Also, it can decrease costs and overcome the biological limitations of the plasmid curve.

Evaluation of cockerel spermatozoa viability and motility by a novel enzyme based cell viability assay.

1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) assay, flow cytometry (FC) or computer-assisted sperm analysis (CASA) were compared. 2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60, and 30 × 106 cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10, 20, 30, 40, 50, 60 min, (2) viability with FC, and (3) motility with CASA. 3. The WST-8 reduction rate was closely correlated with spermatozoa viability and motility. The optimal semen concentration for the WST-8 assay was 120 × 106 cells/ml, and the standard curves for spermatozoa viability and motility predictions, respectively, were yviability60 = 162.8x + 104.96 (R2 = 0.9594) after 60 min of incubation and ymotility40 = 225.09x + 96.299 (R2 = 0.8475) after 40 min of incubation. 4. It was concluded that the WST-8 assay is useful for the practical evaluation of cockerel spermatozoa viability and motility. Compared to FC and CASA, the WST-8 assay does not require expensive and complex instrumentation in the lab. Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa viability and motility at the same time, which all lead it to be efficient and economical for semen quality assessment.

The Inhibitory Effect of Mouse Gastric DNA on Amplification of Helicobacter pylori Genomic DNA in Quantitative PCR

Background: Quantitation of Helicobacter pylori (Hp) in the gastric tissue is essential for assessment of vaccination/therapeutic regimens. Materials & Results: Here, the inhibitory effect of mouse gastric DNA (MgDNA) on amplification of Hp genomic DNA (HpDNA) was evaluated by spiking HpDNA with serial dilutions of MgDNA, which yielded concentrations of >10 ng/µl and >0.63-10 ng/µl of MgDNA, as inhibition and interference zones, respectively. Mice were then inoculated with varying doses of Hp and assessed at the inhibition-free concentration of 0.63 ng/µl. The average Hp copy numbers per microgram of gastric tissue discriminated mice having received high vs. low dose inoculums (p < 0.001). Secondly, Hp copy numbers were quantitated in immunized mice, which demonstrated significantly lower numbers, in reference to controls (p = 0.006). Conclusion: Our method, bypassing the inhibition and/or interference imposed by MgDNA, was able to quantitate gastric tissue-colonizing Hp, segregating mice inoculated with low vs. high doses of Hp, as well as those immunized from controls.

Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients.

AIMS: To improve RT-qPCR with an internal control and a synthetic standard curve to detect HEV in HIV co-infected patients. METHODS AND RESULTS: A single-stranded RNA (ssRNA) and a double-stranded DNA (dsDNA) synthetic curve were designed, compared to the international reference panel for HEV genotypes, and tested to quantify and detect a reference panel for HEV genotypes. The detection limit of the RNA synthetic curve (50 copies per ml) was better than the DNA synthetic curve (100 copies per ml) and the WHO standard curve (250 copies per ml). Then, 280 serum samples from HIV-positive patients were tested for HEV RNA, which was detected in 3·6% of serum samples. The viral load ranged from 2 × 102 copies per ml to 4·78 × 108 copies per ml. HEV IgM/IgG antibodies were not detected in the RNA-positive patients. Sequencing analysis of HEV showed that the virus belongs to genotype 3 (HEV GT3). CONCLUSIONS: Real-time PCR was a useful tool to estimate co-infection with HEV/HIV, even in patients with low viral loads and undetectable anti-HEV IgG and IgM antibodies. SIGNIFICANCE AND IMPACT OF THE STUDY: Hepatitis E virus genotype 3 (HEV GT3) has been associated with silent chronic hepatitis and cirrhosis in HIV-positive subjects worldwide, but there is a lack of data on this co-infection in Brazil.