Analysis of qPCR data by converting exponentially related Ct values into linearly related X0 values

A common method for calculating results from qPCR experiments is the comparative Ct method, also called the 2(-ΔΔCt) method. However, several assumptions are included in the 2(-ΔΔCt) method and standard statistical analyses are not directly applicable. Here, we describe a different method, the X(0) method, for result calculations and statistical analysis from qPCR experiments. The X(0) method differs from the 2(-ΔΔCt) method by introducing a conversion of the exponentially related Ct values into linearly related X(0) values, which represent the amount of starting material in a qPCR experiment. Results calculated by the X(0) method are illustrated for qPCR experiments with technical and biological replicates, including procedures to calculate standard deviations. Incorporation of primer efficiencies in calculations by the X(0) method is also described. Altogether, the X(0) method constitutes a very simple and accurate alternative to the 2(-ΔΔCt) method for result calculations from qPCR data.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

Method for rapid screening of pesticide mineralization in soil

A method has been developed for the analysis of (14)CO(2) evolution from the mineralization of (14)C-labelled organic compounds in soil samples. The new method is less space demanding and substantially cuts down laborious manual work compared to the traditional incubation bottle method used. Furthermore, the use of scintillation cocktail is largely reduced with the new method. In the new method, (14)CO(2) is trapped in filter paper held in the lid of a 20 ml glass vial by surface tension. The trapping solution used is Ca(OH)(2), which fixates CO(2) in the filter paper and the analysis of trapped (14)CO(2) is done using the Cyclone trade mark Storage Phosphor system. The lids are placed in a 32 well holder and exposed to a phosphor screen prior to scanning in a Cyclone trade mark scanner. The new filter method has been tested and compared to results obtained using the traditional method. The results show good agreement but due to a smaller capacity for CO(2) with the filter method compared to the traditional method, the interval between sampling has to be shorter using the filter method when the CO(2) development is high. The detection limits for the filter method is higher compared to the traditional method. With the filter method, the level of radioactivity has to exceed 300 dpm before detection is possible, while the same limit for the traditional method is around 30 dpm. On the other hand, the gas trapping faster and the efficiency is higher with the filter method.     

Microbiological Aspects of Cold Cleaning with an lodophor of Milk Pipeline Installations

Over a period of three years bacteriological comparisons were made of a method of cleaning pipeline milking machines with (1) cold iodophor solution (cold method), (2) a chlorinated alkaline solution at 55-60°C circulated for 10 min (hot method), and (3) acidified boiling water (ABW method). Rinse samples from the entire installations gave the best results with the ABW method. The cold method favoured a psychrotrophic flora, coliform bacteria or lactic streptococci, depending on the part of the milking machine rinsed; the hot method tended to cause a build-up of the heat-resistant flora. In spite of this, the two methods can give acceptable results provided that the installations are correctly designed and constructed.     

Using enhanced development tools offered by analytical Quality by Design to support switching of a quality control method

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE-SDS), with the aim of replacing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a conversion factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy.     

Freezing of bovine embryos: Effects of a one-step addition of 1.4 M glycerol

The effects of a one-step addition of 1.4 M glycerol (method A) upon morphological appearance and developmental capacity of frozen/thawed day 7 bovine embryos were investigated and compared to a standard stepwise addition of 1.0 M glycerol (method B). With method A, the percentage of intact embryos (classified as excellent, good and poor) was 95.3% (61 out of 64) without differences between morulae (96.5%) and blastocysts (94.4%). With method B, the percentage of intact embryos was 83.0% (44 out of 53). The percentage was similar for blastocysts (89.2%) and significantly (p < 0.05) lower for morulae (68.8%) when compared to method A. The percentage of embryos with a damaged zona pellucida was considerably increased with method A (26.6%) when compared to method B (13.2%). The proportion of embryos with excluded blastomeres was similar in both methods (21.9% method A, 17.0% method B). With method A, pregnancy rates after nonsurgical transfer were 51.0% (25 out of 49) and were better than with method B (40.5%; 15 out of 37). Embryos with a damaged zona pellucida resulted in a high pregnancy rate of 66.7% (8 out of 12). A pregnancy rate of 52.9% (10 out of 17) was obtained with embryos showing some excluded blastomeres. Thus, a one-step addition of 1.4 M glycerol facilitates and accelerates the process of embryo cryopreservation and is compatible with high pregnancy rates. Damage of the zona pellucida does not impair further development of frozen/thawed bovine embryos provided blastomeres are intact.     

Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll fluorescence measurements

Day respiration (R(d)) is an important parameter in leaf ecophysiology. It is difficult to measure directly and is indirectly estimated from gas exchange (GE) measurements of the net photosynthetic rate (A), commonly using the Laisk method or the Kok method. Recently a new method was proposed to estimate R(d) indirectly from combined GE and chlorophyll fluorescence (CF) measurements across a range of low irradiances. Here this method is tested for estimating R(d) in five C(3) and one C(4) crop species. Values estimated by this new method agreed with those by the Laisk method for the C(3) species. The Laisk method, however, is only valid for C(3) species and requires measurements at very low CO(2) levels. In contrast, the new method can be applied to both C(3) and C(4) plants and at any CO(2) level. The R(d) estimates by the new method were consistently somewhat higher than those by the Kok method, because using CF data corrects for errors due to any non-linearity between A and irradiance of the used data range. Like the Kok and Laisk methods, the new method is based on the assumption that R(d) varies little with light intensity, which is still subject to debate. Theoretically, the new method, like the Kok method, works best for non-photorespiratory conditions. As CF information is required, data for the new method are usually collected using a small leaf chamber, whereas the Kok and Laisk methods use only GE data, allowing the use of a larger chamber to reduce the noise-to-signal ratio of GE measurements.     

A new statistical method for evaluation of L5178Ytk(+/-) mammalian cell mutation data using microwell method

A statistical method to evaluate data from the mouse lymphoma L5178Y/tk assay (MLA) using microwell method is proposed. This proposed method is designed for data obtained from a single culture protocol instead of the duplicate culture recommended by United Kingdom Environmental Mutagen Society (UKEMS). The proposed method consists of the following three steps: (1) to apply Dunnett type test for identifying clear negative; (2) to apply a Simpson-Margolin procedure for detecting downturn data; and (3) to apply a trend test to evaluate the dose-dependent increase in mutant frequency (MF). The performance of the proposed method was evaluated through a Monte Carlo study and a case study. False positive rates realized in the Monte Carlo study were comparable with the UKEMS method modified for a single culture protocol with the heterogeneity factors being kept at 1.0. False negative rates were less than those of the modified UKEMS method for dose response patterns with a sharp uprise in higher dose groups, whereas, they were comparable for other patterns. The results of evaluating the data from an International Collaborative Study by the proposed method seem comparable with the UKEMS method. The proposed method enables us to evaluate data from the microwell MLA with a single culture protocol.     

Calculating glucose fluxes during meal tolerance test: a new computational approach

A new calculation method is proposed to quantify the endogenous glucose production (EGP), the glucose appearance rate due to meal ingestion (R(a meal)), and the glucose disposal (R(d)) during a three-tracer study design. The method utilizes the maximum likelihood theory combined with a regularization method to achieve a theoretically coherent computational framework. The method uses the two-compartment formulation of the glucose kinetics. Instead of assuming smoothness of unlabeled and labeled glucose concentrations, the method assumes that the EGP, the R(a meal), and the fractional glucose clearance are smooth, increasing plausibility of their individual estimates. The method avoids transformation of the measurement errors, which may skew the estimates of the EGP, R(a meal), and R(d) with the traditional approach. Finally, the sequential nature of the calculations is replaced by calculating the EGP, R(a meal), and R(d) in "one go" to avoid the propagation of the errors from the EGP and R(a meal) into R(d). An example study is shown demonstrating the utility of the approach. A better performance of the new method is demonstrated in a simulation study.     

The relative efficiency of the Hardy-Weinberg equilibrium-likelihood and the conditional on parental genotype-likelihood methods for candidate-gene association studies.

Selecting a control group that is perfectly matched for ethnic ancestry with a group of affected individuals is a major problem in studying the association of a candidate gene with a disease. This problem can be avoided by a design that uses parental data in place of nonrelated controls. Schaid and Sommer presented two new methods for the statistical analysis using this approach: (1) a likelihood method (Hardy-Weinberg equilibrium [HWE] method), which rests on the assumption that HWE holds, and (2) a conditional likelihood method (conditional on parental genotype [CPG] method) appropriate when HWE is absent. Schaid and Sommer claimed that the CPG method can be more efficient than the HWE method, even when equilibrium holds. It can be shown, however that in the equilibrium situation the HWE method is always more efficient than the CPG method. For a dominant disease, the differences are slim. But for a recessive disease, the CPG method requires a much larger sample size to achieve a prescribed power than the HWE method. Additionally, we show how the relative risks for the various candidate-gene genotypes can be estimated without relying on iterative methods. For the CPG method, we represent an asymptotic power approximation that is sufficiently precise for planning the sample size of an association study.     

Field Evaluation of Two Colorimetric Coliphage Detection Methods

Two new methods for coliphage detection, a colorimetric agar-based (CAB) method and a liquid colorimetric presence-absence (LCPA) method, were compared to the coliphage method proposed by the American Public Health Association (APHA; Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association, Washington, D.C., 1992). Both new methods are based on the induction of β-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate which produces a colored reaction product. Ninety split water samples from four different sources were tested. A total of 52 samples were positive by the CAB method, 52 were positive by the LCPA method, and 53 were positive by the APHA method. Results indicated that (i) the CAB and LCPA methods were as sensitive in coliphage detection as the APHA method, (ii) both the CAB and LCPA methods were easier to read and interpret than the APHA method, and (iii) the CAB method detected more coliphages in a positive sample than the APHA method in two of the four types of water sources. Importantly, the rapid and simple LCPA method was as reliable and sensitive as either of the two agar-based methods in coliphage detection.     

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.     

A novel method for constructing gene-targeting vectors

We developed a simple and rapid method for constructing knockout vectors using inverse-PCR (IPCR). The method consists of three steps: (i) digestion of a target bacterial artificial chromosome with several restriction enzymes (six-base cutters) followed by self-ligation; (ii) IPCR using circular DNAs as templates and two primers which are oriented in opposite directions; and (iii) cloning into a vector containing a positive selection marker, which results in a typical replacement knockout vector. We successfully targeted three mouse genes including the HPRT gene using this method. Compared with the conventional method, this method requires much less time (no more than 3 weeks). Notably, this method requires only small amounts of sequence information (several hundred base pairs such as is available from expressed sequence tags) and can be extended to a systematic mass production of targeting vectors applicable to many organisms, including yeast.     

Approximating identity-by-descent matrices using multiple haplotype configurations on pedigrees

Identity-by-descent (IBD) matrix calculation is an important step in quantitative trait loci (QTL) analysis using variance component models. To calculate IBD matrices efficiently for large pedigrees with large numbers of loci, an approximation method based on the reconstruction of haplotype configurations for the pedigrees is proposed. The method uses a subset of haplotype configurations with high likelihoods identified by a haplotyping method. The new method is compared with a Markov chain Monte Carlo (MCMC) method (Loki) in terms of QTL mapping performance on simulated pedigrees. Both methods yield almost identical results for the estimation of QTL positions and variance parameters, while the new method is much more computationally efficient than the MCMC approach for large pedigrees and large numbers of loci. The proposed method is also compared with an exact method (Merlin) in small simulated pedigrees, where both methods produce nearly identical estimates of position-specific kinship coefficients. The new method can be used for fine mapping with joint linkage disequilibrium and linkage analysis, which improves the power and accuracy of QTL mapping.     

A method for determining the apoplastic water volume of conifer needles

A method for direct estimation of percentage apoplastic water volume (% APO) in conifer needles is described. The method presented here, and designated the pressure-needle (P-N) method, measures the relative water content of the needles to develop a curve similar to the pressure-volume (P-V) curve. P-V and P-N curves were developed for Picea pungens Engelm. cv. Hoopsi, Pinus sylvestris L., Abies gradis (Dougl.) L., and Pseudotsuga menziesii (Mirb) Franco. The % APO estimated by the two procedures varied as much as 2-fold, while other parameters were similar. The P-V method generated consistently higher and more variable % APO than the P-N method, due to the inclusion of the apoplastic water of the stem in the P-V method. For conifers, the P-N method offers a more accurate and precise method for determining % APO.     

啮齿动物的巢区面积估算法

巢区(Home range)是动物在其巢附近进行取食、生殖、育幼等日常活动的区域(Burt 1940)。标志流放法是应用最广的调查啮齿动物巢区的方法,尤其是按方格式布笼。但对同一野外调查结果,由于估算方法不同,巢区估算值相差很大,并且至今尚无学者提出一致公认的估算法。1980年5-10月,我们在青海省门源县的高寒草甸生态系统定位站调查根田鼠的巢区,按多种估算法对同一批实际调查结果估计了巢区面积,并对结果进行了分析比较,检查其特点和优缺点,并提出修正平均值法,作为我们今后讨论根田鼠巢区动态的基础。 对方格式布笼的调查结果进行巢区的估算方法,基本上分二大类,图形法和概率性模型法。图形法是按照捕点分布划出巢区,并直接求出巢区面积,如最小面积法,包括或不包括周边地带法,最大距离法和复合散布图法。     

Genotyping and annotation of Affymetrix SNP arrays

In this paper we develop a new method for genotyping Affymetrix single nucleotide polymorphism (SNP) array. The method is based on (i) using multiple arrays at the same time to determine the genotypes and (ii) a model that relates intensities of individual SNPs to each other. The latter point allows us to annotate SNPs that have poor performance, either because of poor experimental conditions or because for one of the alleles the probes do not behave in a dose–response manner. Generally, our method agrees well with a method developed by Affymetrix. When both methods make a call they agree in 99.25% (using standard settings) of the cases, using a sample of 113 Affymetrix 10k SNP arrays. In the majority of cases where the two methods disagree, our method makes a genotype call, whereas the method by Affymetrix makes a no call, i.e. the genotype of the SNP is not determined. By visualization it is indicated that our method is likely to be correct in majority of these cases. In addition, we demonstrate that our method produces more SNPs that are in concordance with Hardy–Weinberg equilibrium than the method by Affymetrix. Finally, we have validated our method on HapMap data and shown that the performance of our method is comparable to other methods.     

Validation of a questionnaire method for estimating extent of menstrual blood loss in young adult women.

The objective of this study was to validate two indirect methods for estimating the extent of menstrual blood loss against a reference method to determine which method would be most appropriate for use in a population of young adult women. Thirty-two women aged 18 to 29 years (mean +/- SD; 22.4 +/- 2.8) were recruited by poster in Dunedin (New Zealand). Data are presented for 29 women. A recall method and a record method for estimating extent of menstrual loss were validated against a weighed reference method. Spearman rank correlation coefficients between blood loss assessed by Weighed Menstrual Loss and Menstrual Record was rs = 0.47 (p = 0.012), and between Weighed Menstrual Loss and Menstrual Recall, was rs = 0.61 (p = 0.001). The Record method correctly classified 66% of participants into the same tertile, grossly misclassifying 14%. The Recall method correctly classified 59% of participants, grossly misclassifying 7%. Reference method menstrual loss calculated for surrogate categories demonstrated a significant difference between the second and third tertiles for the Record method, and between the first and third tertiles for the Recall method. The Menstrual Recall method can differentiate between low and high levels of menstrual blood loss in young adult women, is quick to complete and analyse, and has a low participant burden.     

A simple and rapid method for calculating identity-by-descent matrices using multiple markers

A fast, partly recursive deterministic method for calculating Identity-by-Descent (IBD) probabilities was developed with the objective of using IBD in Quantitative Trait Locus (QTL) mapping. The method combined a recursive method for a single marker locus with a method to estimate IBD between sibs using multiple markers. Simulated data was used to compare the deterministic method developed in the present paper with a stochastic method (LOKI) for precision in estimating IBD probabilities and performance in the task of QTL detection with the variance component approach. This comparison was made in a variety of situations by varying family size and degree of polymorphism among marker loci. The following were observed for the deterministic method relative to MCMC: (i) it was an order of magnitude faster; (ii) its estimates of IBD probabilities were found to agree closely, even though it does not extract information when haplotypes are not known with certainty; (iii) the shape of the profile for the QTL test statistic as a function of location was similar, although the magnitude of the test statistic was slightly smaller; and (iv) the estimates of QTL variance was similar. It was concluded that the method proposed provided a rapid means of calculating the IBD matrix with only a small loss in precision, making it an attractive alternative to the use of stochastic MCMC methods. Furthermore, developments in marker technology providing denser maps would enhance the relative advantage of this method.     

A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H(2)O(2) level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC(50)) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC(50) by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H(2)O(2) levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.