Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.     

Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription—Polymerase chain reaction

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.     

竞争性RT-PCR测定法及BMP-2mRNA的定量检测

建立竞争性 PCR方法 ,以期用于转录水平基因表达的定量研究 .以检测大鼠颅骨骨细胞总RNA中骨形态发生蛋白 - 2 ( BMP- 2 ,bone morphogenetic protein- 2 ) m RNA含量为例 ,构建大鼠BMP- 2基因的竞争模板 ,以之为内对照进行竞争性 PCR.PCR反应结束后 ,电泳、拍照 ,扫描所扩增条带密度 ,作回归方程 .根据回归方程 ,计算出正常 7d Wistar大鼠颅骨总 RNA中 BMP- 2 m RNA含量为 1 .1 2 5amol/μg RNA.结果显示 ,成功构建了大鼠 BMP- 2基因的竞争模板 ,建立了可以测定大鼠颅骨骨细胞总 RNA中 BMP- 2 m RNA含量的竞争性 PCR方法 .     

Competitive PCR for precise nucleic acid quantification

The exact quantification of tiny amounts of nucleic acids in biological samples continues to remain a requirement in both the experimental and the diagnostic laboratory. Competitive PCR involves the coamplification of a target DNA sample with known amounts of a competitor DNA that shares most of the nucleotide sequence with the target; in this way, any predictable or unpredictable variable affecting PCR amplification has the same effect on both molecular species. Competitive PCR therefore permits the quantification of the absolute number of target molecules in comparison to the amount of competitor DNA. Although requiring intensive post-PCR manipulation, the accuracy of competitive PCR by far exceeds that of any other quantitative PCR procedure, including real-time PCR. This protocol covers all stages in the competitive PCR and RT-PCR methods, from the design and construction of competitor molecules, and the competitive PCR itself, to the analysis of data and quantification of target DNA. Once the correct primers are available, the protocol can be completed in about 24 h.     

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.     

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.     

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.     

一种用少量标本快速构建老年性白内障消减cDNA文库的方法

为从少量标本中获得含较多大片段的、高质量的老年性白内障消减cDNA文库,利用磁珠分离、生物素标记的改良消减杂交法获得差异cDNA,利用选择性PCR法扩增其中大片段差异cDNA,从而成功构建老年性白内障消减cDNA文库.在文库中随机挑取的22个克隆中,1 000 bp以上的片段有7个,占31.8%,750 bp以上有15个,占68.2%.所得cDNA片段较大,可以满足下一步研究需要.改良消减杂交法结合选择性PCR法可以从少量标本中快速有效地获得大片段高质量的消减cDNA文库.     

Adapter-tagged competitive PCR (ATAC-PCR) – a high-throughput quantitative PCR method for microarray validation

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.     

Adapter-tagged competitive PCR (ATAC-PCR)--a high-throughput quantitative PCR method for microarray validation

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.     

Quantification of mRNA Levels by Fluorescently Labelled Reversde Transcription Competitive PCR

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Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.     

Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes

Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.     

早老性痴呆大脑cDNA文库构建及目的基因克隆

 取临床确诊为早老性痴呆 (Alzheimer’sdisease ,AD)患者的大脑组织 ,应用磁珠法直接提取mRNA ,电泳检测其质量 .经逆转录合成双链cDNA后 ,用碱性凝胶电泳检测其大小在 0 .2～ 9.0kb范围 ,主要集中在 1.0～ 2 .0kb之间 .层析除去多余的adaptors ,收集大于 4 0 0bp的cDNA片段 ,与载体pYESTrp2连接 ,经电转化后 ,得到克隆总数为 5.1× 10 5的AD病人大脑cDNA文库 .用PCR技术从该文库中扩增得到小肠三叶因子 (intestinaltrefoilfactor ,ITF) [1] 和神经生长抑制因子 (growthin hibitoryfactor,GIF) [2 ] 的cDNA编码区 .研究表明 ,所构建的cDNA文库质量较高 ,可广泛用于AD病研究工作 .同时 ,将所克隆的GIF编码区插入到载体pHybLex Zeo上 ,构建成带饵基因的质粒 ,为进一步通过酵母双杂交方法搜寻与GIF相互作用的神经因子提供了必要条件