Subcellular localization of HCV core protein regulates its ability for p53 activation and p21 suppression

An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.     

Identifying and quantifying sources of variation in microarray data using high-density cDNA membrane arrays.

Microarray experiments involve many steps, including spotting cDNA, extracting RNA, labeling targets, hybridizing, scanning, and analyzing images. Each step introduces variability, confounding our ability to obtain accurate estimates of the biological differences between samples. We ran repeated experiments using high-density cDNA microarray membranes (Research Genetics Human GeneFilters Microarrays Version I) and 33P-labeled targets. Total RNA was extracted from a Burkitt lymphoma cell line (GA-10). We estimated the components of variation coming from: (1) image analysis, (2) exposure time to PhosphorImager screens, (3) differences in membranes, (4) reuse of membranes, and (5) differences in targets prepared from two independent RNA extractions. Variation was assessed qualitatively using a clustering algorithm and quantitatively using a version of ANOVA adapted to multivariate microarray data. The largest contribution to variation came from reusing membranes, which contributed 38% of the total variation. Differences in membranes and in exposure time each contributed about 10%. Differences in target preparations contributed less than 5%. The effect of image quantification was negligible. Much of the effect from reusing membranes was attributable to increasing levels of background radiation and can be reduced by using membranes at most four times. The effects of exposure time, which were partly attributable to variation in the scanning process, can be minimized by using the same exposure time for all experiments.     

Primer extension studies on α-amylase mRNAs in barley aleurone. I. Characterization and quantification of the transcripts

Primer extension was used to characterize alpha-amylase mRNAs from aleurone tissue of barley (Hordeum vulgare L. cv. Himalaya) grains. Two synthetic oligonucleotides, specific for the low-pI and high-pI alpha-amylase groups, were used as primers for synthesis of cDNA from total RNA preparations. Between them, these two oligonucleotides appear to account for all major alpha-amylase mRNAs as judged by hybrid-arrested translation of alpha-amylase mRNAs in a cell-free system. Reconstruction experiments indicated that the levels of extended primers (determined by scintillation counting) were directly proportional to the level of input mRNA over a wide range. This indicates that the technique is suitable for quantification of relative levels of individual alpha-amylase from approximately 2% to 100% of maximal levels. The nucleotide sequences of extended primers defined two different alpha-amylase mRNAs in each of the low-pI and high-pI groups, and possibly a third mRNA in the high-pI group.     

Characterization of variability in large-scale gene expression data: implications for study design

Large-scale gene expression measurement techniques provide a unique opportunity to gain insight into biological processes under normal and pathological conditions. To interpret the changes in expression profiles for thousands of genes, we face the nontrivial problem of understanding the significance of these changes. In practice, the sources of background variability in expression data can be divided into three categories: technical, physiological, and sampling. To assess the relative importance of these sources of background variation, we generated replicate gene expression profiles on high-density Affymetrix GeneChip oligonucleotide arrays, using either identical RNA samples or RNA samples obtained under similar biological states. We derived a novel measure of dispersion in two-way comparisons, using a linear characteristic function. When comparing expression profiles from replicate tests using the same RNA sample (a test for technical variability), we observed a level of dispersion similar to the pattern obtained with RNA samples from replicate cultures of the same cell line (a test for physiological variability). On the other hand, a higher level of dispersion was observed when tissue samples of different animals were compared (an example of sampling variability). This implies that, in experiments in which samples from different subjects are used, the variation induced by the stimulus may be masked by non-stimuli-related differences in the subjects' biological state. These analyses underscore the need for replica experiments to reliably interpret large-scale expression data sets, even with simple microarray experiments.     

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.     

Variability in messenger RNA levels in human umbilical vein endothelial cells of different lineage and time in culture

Summary Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plaminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.     

表达谱基因芯片的可靠性验证分析

cDNA芯片是一项新兴的能评估检测全范围mRNA表达水平变化的技术。通过同种组织RNA自身比较实验及不同组织RNA的差异分析实验对cDNA芯片实验的重复性进行检验,利用相关系数(correlation coefficient,R)、变异系数(coefficient of variation,CV)和假阳性率(false positiver ate,FPR)分析eDNA芯片数据的可靠程度,对cDNA芯片实验数据作了整体的评估。结果证实,该芯片系统得到的cDNA表达谱数据相关系数一般大于0．9,平均变异系数15％左右,假阳性率控制在3％以内。还提出一致率(consistence rate,CR)的概念,作为衡量cDNA芯片系统重复性的新参数,同时阐述了该参数优于目前常用的相关系数及变异系数的特点。另外,通过比较芯片制备中点样浓度、mRNA和总RNA以及不同批次芯片和不同标记过程对实验的影响,来分析芯片数据的系统误差来源。并提出重复两次实验,可以克服绝大部分实验系统引入的假阳性。     

High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.

To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.     

Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.     

成纤维细胞生长因子21的研究进展

以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用。分别采集无芒隐子草幼苗自然干旱0、2、4、6、8、10d以及恢复浇水1、4d的叶片和根系材料,相对定量采用2 -△△CT法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化。相对定量2 -△△CT法结果显示,CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍。绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4～6d和恢复浇水后1～4d,CsSAMDC基因在叶和根组织中的表达较对照降低。相对定量和绝对定量在CsSAMDC基因表达研究中显示相对一致的趋势。     

A color composite technique for detecting root dynamics of barley (Hordeum vulgare L.) from minirhizotron images

Quantification of root dynamics by destructive methods is confounded by high coefficients of variation and loss of fine roots. The minirhizotron technique is non-destructive and allows for sequential root observations to be made at the same depth in situ. Observations can be stored on video tape which facilitates data handling and computer-aided image processing. A color composite technique using digital image analyses was adapted in this study to detect barley root dynamics from sequential minirhizotron images. Plants were grown in the greenhouse in boxes (80 × 80 × 75 cm) containing soil from a surface horizon of a Typic Cryoboroll. A minirhizotron was installed at a 45°C angle in each box. Roots intersecting the minirhizotron were observed and video-recorded at tillering, stem extension, heading, dough and ripening growth stages. The images from a particular depth were digitized from the analog video then registered to each other. Discrimination of roots from the soil matrix gave quantitative estimates of root appearance and disappearance. Changes in root appearance and disappearance were detected by assigning a separate primary color (red, green, blue) to selected growth stages, then overlaying the images to create red-green and red-green-blue color composites. The resulting composites allowed for a visual interpretation and quantification of barley root dynamics in situ.     

A quantitative proteomic analysis of light adaptation in a globally significant marine cyanobacterium Prochlorococcus marinus MED4

In this study, we conducted biological and technical replicate proteomic experiments using isobaric tags for relative and absolute quantification (iTRAQ), to elucidate the light adaptation strategies of Prochlorococcus marinus MED4. The MED4 strain is adapted to an oceanic environment characterized by low nutrient levels, and ever-changing light intensities. Approximately 11% of the proteome was identified, with an average coefficient of variation of iTRAQ quantification values of 0.15. Fifteen proteins were deemed to be statistically and significantly differentially expressed in changing light intensities, particularly the down-regulation of photosystem-related proteins, and the up-regulation of the stress-related chaperone GroEL in high light compared to low light.