运用多种策略改良差异显示PCR

目的：改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法：①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果：减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论：改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。     

Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones--Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin--which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因

为筛选血管外膜成纤维细胞（adventitial fibroblast,AF）与肌成纤维细胞（myofibroblast,MF）间表型转化有关的基因,实验建立了大鼠胸主动脉AF和MF两种细胞模型,用差异显示聚合酶链反应（DD－PCR）技术获得表达差异片段,对差异片段进行克隆和测序分析,并用定量PCR和Northern blot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白（osteopontin,OPN）对AF迁移的影响。结果表明,两种表型细胞存在明显的基因表达差异,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位5（NADH dehydrogenase subunit 5,Nd5）基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northern blot证实。此外还有4个表达序列标志（expressed sequence-tag,EST）在GenBank中未查到同源序列。反义OPN寡脱氧核甘酸可抑制AF的迁移活动。结果提示,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。     

Screening for ribosomal-based false positives following prokaryotic mRNA differential display

Differential display (DD) and the closely related RNA arbitrarily primed PCR (RAP-PCR) have become the molecular tools of choice for identifying and isolating differentially expressed genes in both eukaryotic and prokaryotic systems. However, one of the current drawbacks of both techniques is the high number of false positives generated. In prokaryotic applications, the many false positive typically generated by DD are subsequently identified as rRNAs because of their greater abundance compared to mRNAs. To circumvent this problem, full-length 16S and 23S rDNA probes, derived from Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1, respectively, were used as a prescreening approach to discriminate between those bands, which appear to be differentially expressed mRNAs, but in fact are rRNAs, following prokaryotic mRNA DD.     

致心律失常型右室心肌病引起的心力衰竭分子标志物的筛选

目的：筛选致心律失常型右室心肌病引起心力衰竭的分子标志物。方法：从本院的心脏病组织库中挑选5例病理诊断明确和各方面资料比较齐全的致心律失常型右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行全基因组表达芯片的比较研究。提取致心律失常型右室心肌病的左心室组织RNA,同时提取正常对照心脏相应部位的RNA。应用晶芯人类全基因组寡核苷酸微阵列基因表达谱芯片(含有人类基因35,000个),筛选致心律失常型右室心肌病引起的心力衰竭基因表达谱的改变。应用实时定量荧光反转录聚合酶链式反应(real-time RT-PCR)验证致心律失常型右室心肌病引起的心力衰竭基因表达改变的真实性和准确性。 结果：应用基因表达芯片研究方法共筛选出78个差异表达基因,其中有35个基因在致心律失常型右室心肌病引起的心力衰竭中表达升高,而另有43个基因表达降低。其中变化较多的基因属于与代谢相关的基因。对其中36个差异表达基因应用real-time RT-PCR的方法进行了验证,差异表达基因的准确性在75%,并首次报告了心钠素在致心律失常型右室心肌病引起的心力衰竭中表达明显升高。结论：本研究在世界上首次应用基因表达芯片的方法,观察了致心律失常型右室心肌病引起的心力衰竭基因表达谱的改变,为致心律失常型右室心肌病引起的心力衰竭分子机制的阐明和寻找疾病特异的分子标志物,以用于鉴别诊断、判断病情和预后及指导个性化治疗奠定了基础。     

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.     

Differential mRNA fingerprinting by preferential amplification of coding sequences

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.     

Identification of genes associated with tumorigenesis of retinoblastoma by microarray analysis

There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.     

Postdifferential display: parallel processing of candidates using small amounts of RNA

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.     

The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display

In many applications, an understanding of differentially expressed genes in different tissues, or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential, display [DDR-PCR] has shown, that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.     

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Δ¹³C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE. Electronic Supplementary Material Supplementary material is available for this article at