共查询到20条相似文献,搜索用时 31 毫秒
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Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level 总被引:1,自引:0,他引:1
Background
Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies. 相似文献3.
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S. Schlosser G. Gröger O. Holfeld H. Seliger 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):1515-1522
Abstract Using appropriately designed primers we amplified a 783 bp frag ment of the triple helical portion of human α1-collagen (I) by PCR methods. The expression of this biosynthetic collagen gene and a modified one containing cell adhesion sequences was carried out in a prokaryotic (E. coli) and a eukaryotic (baculovirus) system. The expression products were purified by affinity chromatography. 相似文献
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Minute tissue samples or single cells increasingly provide the starting material for gene expression profiling, which often requires RNA amplification. Although much effort has been put into optimizing amplification protocols, the relative abundance of RNA templates in the amplified product is frequently biased. We applied a T7 polymerase-based technique to amplify RNA from two tissues of a cichlid fish and compared expression levels of unamplified and amplified RNA on a cDNA microarray. Amplification bias was generally minor and comprised features that were lost (1.3%) or gained (2.5%) through amplification and features that were scored as regulated before but unregulated after amplification (4.2%) or vice versa (19.5%). We examined 10 sequence-specific properties and found that GC content, folding energy, hairpin length and number, and lengths of poly(A) and poly(T) stretches significantly affected RNA amplification. We conclude that, if RNA amplification is used in gene expression studies, preceding experiments controlling for amplification bias should be performed. 相似文献
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Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification 总被引:2,自引:0,他引:2
Kaposi-Novak P Lee JS Mikaelyan A Patel V Thorgeirsson SS 《BioTechniques》2004,37(4):580, 582-586, 588
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Xiang CC Kozhich OA Chen M Inman JM Phan QN Chen Y Brownstein MJ 《Nature biotechnology》2002,20(7):738-742
DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers. 相似文献
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Summary. Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses. 相似文献
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A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies. 相似文献
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Fan JB Chen J April CS Fisher JS Klotzle B Bibikova M Kaper F Ronaghi M Linnarsson S Ota T Chien J Laurent LC Loring JF Nisperos SV Chen GY Zhong JF 《PloS one》2012,7(2):e30794