Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells

Background  

Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets.     

Human caspases in vitro: expression, purification and kinetic characterization

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.     

State‐of‐the‐art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β‐tubulin and β‐actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large‐scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem‐mass‐spectrometry data from 27 human tissues, and suggest 20 ubiquitously‐ and constitutively‐expressed, putative internal‐reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.     

Chemiluminescence quantitative immunohistochemical determination of MRP2 in liver biopsies.

Evaluation of protein expression in tissues and cells by electrophoretic and blotting techniques or by the quantification of the mRNA coding for the target protein is a common procedure in biochemistry research and clinical diagnoses. In this article, an alternative approach, based on an immunohistochemical procedure with chemiluminescent imaging detection, is described. The assay exploited the peculiar characteristics of the chemiluminescent detection of enzyme labels (high sensitivity and specificity, low background, easy quantification of the signal) for performing the direct, simple, and rapid quantitative evaluation of protein expression in tissues. When applied to the study of the levels of MRP2, a member of the human multidrug resistance-associated protein family, in samples obtained from formalin-fixed, paraffin-embedded liver biopsies, it allowed the reliable evaluation of the protein content of the tissue. Moreover, the analysis of clinical samples from patients with primary biliary cirrhosis under therapy with ursodeoxycholic acid gave results in line with those, previously reported in the literature, obtained with conventional protein expression analysis techniques.     

On the reliability of peptide nonplanarity seen in ultra‐high resolution crystal structures

Ultra‐high resolution protein crystal structures have been considered as relatively reliable sources for defining details of protein geometry, such as the extent to which the peptide unit deviates from planarity. Chellapa and Rose (Proteins 2015; 83:1687) recently called this into question, reporting that for a dozen representative protein structures determined at ～1 Å resolution, the diffraction data could be equally well fit with models restrained to have highly planar peptides, i.e. having a standard deviation of the ω torsion angles of only ～1° instead of the typically observed value of ～6°. Here, we document both conceptual and practical shortcomings of that study and show that the more tightly restrained models are demonstrably incorrect and do not fit the diffraction data equally well. We emphasize the importance of inspecting electron density maps when investigating the agreement between a model and its experimental data. Overall, this report reinforces that modern standard refinement protocols have been well‐conceived and that ultra‐high resolution protein crystal structures, when evaluated carefully and used with an awareness of their levels of coordinate uncertainty, are powerful sources of information for providing reliable information about the details of protein geometry.     

MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain

The most popular strategy for normalization of RT-qPCR data involves presenting them in comparison with expression of “housekeeping” genes. However, the required stable expression of the control genes is not always achievable. As an alternative, we used ribonucleoprotein phage particles as an exogenous internal control and demonstrated that this type of normalization provides a simple and reliable method for quantification in RT-qPCR experiments. Using phage-based normalization, we analyzed mRNA levels of three popular housekeeping genes coding β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein L30 and showed high variability in their expression patterns during rat brain development, indicating that they should not be used as controls in gene expression studies of the developing brain either individually or in combination. Using phage-based controls, we showed interstrain differences and age-related changes in the expression of genes involved in proteoglycan biosynthesis and degradation in developing brain of senescenceaccelerated OXYS rats and control Wistar rats.     

Quantitative Evaluation of Aldo-keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo–keto reductases (AKRs) in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measurements of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring (MRM) of mass spectrometry (MS) to examine the protein expression of AKRs in five different hepatic cell lines. These include one relatively normal hepatic cell line, L-02, and four hepatocellular carcinoma (HCC) cell lines, HepG2, HuH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HuH7. Similar observations were obtained through MRM. Different from HuH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distribution of AKR1C proteins is likely to be associated with liver tumorigenesis, and the AKR expression status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HuH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein family at both mRNA and protein levels were feasible using current techniques.     

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.     

实时荧光定量PCR的发展和数据分析

实时荧光定量PCR技术是基因时代一项用于检测mRNA的常用技术,是临床检测和基础研究中不可缺少的重要研究方法,包括绝对定量PCR和相对定量PCR。该技术的特点是可以减少PCR后操作,在比较不同浓度的mRNA方面具有非常宽的动力学范围。我们就目前实时荧光定量PCR的发展及数据的分析进行综述。     

EF1α and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells

Background  

RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells.