鳜鱼基因表达转录分析中的内参选择比较

目前基因表达的转录分析多采用单一或多个看家基因作为内参来校正目的基因的表达量。该实验以鳜鱼6个不同组织和5个不同胚胎发育阶段为研究对象,应用实时荧光定量PCR技术,观察了GAPDH、β-actin和18S rRNA三个看家基因mRNA水平的表达情况。geNorm统计分析表明,胚胎发育阶段β-actin表达最为稳定;不同的组织样品间,GAPDH表达最为稳定;而18S rRNA 的表达在不同的发育阶段不稳定。当利用多基因作为内参时,使用两个最稳定表达的看家基因即可对目的基因的表达进行准确校正。该结果证实了基因表达转录分析中内参基因选择的必要性,同时为鳜鱼等鱼类基因表达分析时内参基因的选择提供有价值的参考     

小鼠基因转录表达分析中内参基因的优选

目的 建立小鼠基因转录表达分析中内参基因的选择方法.方法 以C57BL/6J和C3H/HeJ两个品系3个不同组织及2个不同发育阶段为研究对象,应用反转录实时定量PCR技术,评价GAPDH(glyceraldehyde-3-phosphate dehydrogenase)、HPRTl(hypoxanthine phosphoribosyl transferase)、B2M(β2-microglobulin)、PPIA(peptidylprolyl isomerase A)、ACTB(Actin-beta)和18S rRNA(18S ribosomal RNA)等6个看家基因在下丘脑、垂体与卵巢中mRNA水平的表达稳定性.结果 GeNorm统计分析表明,GAPDH和HPRT1表达最为稳定,PPIA等次之,B2M在不同组织和发育阶段中都几乎无表达.结论 成功筛选到GAPDH和HPRT1两个稳定表达的看家基因,证实了小鼠基因表达转录分析中内参基因选择的必要性和可行性.     

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Quantification of PCR products by phosphate measurement

Various techniques for quantification of PCR are available. Most frequently, the densitometric intensities of ethidium bromide-stained PCR products separated in gels are compared after normalizing to the levels of housekeeping gene products such as β-actin. More precise, but extremely time consuming, is the technique of competitive PCR. Newer methods, such as tracking amplification in real-time, have high start-up and maintenance costs (e.g., TaqMan, Applied Biosystems; LightCycler, Roche; I-Cycler, Bio-Rad). Here, I describe an alternative, simple technique to quantify PCR products by determining the entire phosphate released during PCR. The method can be performed using common laboratory equipment, and the reagents needed are extremely cheap. The method is validated by measuring the induction of inducible nitric oxide synthase gene expression in cell culture and comparing the results with data obtained by LightCycler experiments and RNase protection assays.     

Peptidylpropyl isomerase B (PPIB): a suitable reference gene for mRNA quantification in peripheral whole blood

Quantitative real-time RT-PCR is a very powerful technique for measuring gene expression at the mRNA level. In order to compare mRNA expression in different experimental or clinical conditions, expression of a target gene has to be normalized to an appropriate internal standard, which is generally a housekeeping gene. In our study, we have tested several housekeeping genes in peripheral whole blood of healthy volunteers and patients suffering from inflammatory diseases. A first analysis of 91 samples illustrated that the mRNA expression of peptidylpropyl isomerase B (PPIB) encoding for cyclophilin B protein, is more stable than beta actin and glyceraldehyde-3-phosphate dehydrogenase, which are both commonly selected as internal standard. Among the three genes tested, beta actin displayed the highest inter-sample variation of expression. The constancy of PPIB mRNA expression was further confirmed by 214 additional samples. In conclusion, we showed that PPIB, in contrast to beta actin and glyceraldehyde-3-phosphate dehydrogenase, is a suitable housekeeping gene in human peripheral blood.     

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi-quantitative PCR

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-gamma. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0-4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-gamma; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.     

应用SYBR实时荧光定量Real—TimePCR法检测人宫颈癌MCPH1 mRNA表达

目的 应用SYBR实时荧光定量RT-PCR法检测MCPH1/BRIT1 mRNA的表达.方法 提取人宫颈癌总RNA,经逆转录PCR获得靶基因（MCPH1）及管家基因（GAPDH）的CDNA,采用SYBR Green 荧光实时定量法检测,以GAPDH基因作为内参,计算各组MCPH1 mRNA的相对表达量.结果 在31例宫颈癌标本中,MCPH1基因mRNA的表达,19例癌比正常低,1 例癌比正常高,其余标本无统计学意义;6例癌比癌旁低,1例癌比癌旁高,其余无统计学意义.结论 利用SYBR实时荧光定量RT-PCR检测出人宫颈癌中MCPH1基因mRNA的表达下调,为进一步研究MCPH1在宫颈癌中的作用及功能奠定了基础.     

Similarity queries for temporal toxicogenomic expression profiles

We present an approach for answering similarity queries about gene expression time series that is motivated by the task of characterizing the potential toxicity of various chemicals. Our approach involves two key aspects. First, our method employs a novel alignment algorithm based on time warping. Our time warping algorithm has several advantages over previous approaches. It allows the user to impose fairly strong biases on the form that the alignments can take, and it permits a type of local alignment in which the entirety of only one series has to be aligned. Second, our method employs a relaxed spline interpolation to predict expression responses for unmeasured time points, such that the spline does not necessarily exactly fit every observed point. We evaluate our approach using expression time series from the Edge toxicology database. Our experiments show the value of using spline representations for sparse time series. More significantly, they show that our time warping method provides more accurate alignments and classifications than previous standard alignment methods for time series.     

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.