Abstract：
:Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Scherer A,Krause A,Walker JR,Sutton SE,Serón D,Raulf F,Cooke MPdoi
10.2144/03343rr01subject
Has Abstractpub_date
2003-03-01 00:00:00pages
546-50, 552-4, 556issue
3eissn
0736-6205issn
1940-9818journal_volume
34pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract：:Several techniques were evaluated for the quantitation of the total protein content of an IgG2a monoclonal antibody, KS1/4, and its deacetylvinblastine (DAVLB) conjugate. The UV assay is rapid, but it requires an extensive calibration of the response factor, and impurities may cause a high bias. Amino acid analysis (A...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1988-11-01 00:00:00
abstract：:The trend toward high-throughput techniques in molecular biology and the explosion of online scientific data threaten to overwhelm the ability of researchers to take full advantage of available information. This problem is particularly severe in the rapidly expanding area of gene expression experiments, for example, t...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/99276bc03
更新日期：1999-12-01 00:00:00
abstract：:The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converte...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112757
更新日期：2008-02-01 00:00:00
abstract：:We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112629
更新日期：2007-12-01 00:00:00
abstract：:DNAdraw is a Macintosh program designed to prepare DNA and protein data for publication. In additional to providing standard ways of highlighting data, e.g., fonts, styles and shading, DNAdraw has special features for formatting sequence data and for handling aligned sequence data. Output for the program is to a PostS...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1995-06-01 00:00:00
abstract：:In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112476
更新日期：2007-06-01 00:00:00
abstract：:We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosp...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1995-01-01 00:00:00
abstract：:We have utilized 5-bromo-2'deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization. Br-dUTP was used as a substitute...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1996-07-01 00:00:00
abstract：:The purpose of this research was to compare three bioreactor systems for the pilot-scale production of monoclonal antibodies (MAbs). We needed to produce gram quantities of murine MAbs against human prostatic acid phosphatase for use in fragmentation, radiolabeling and in vivo radio-imaging studies. The stable hybrido...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1993-10-01 00:00:00
abstract：:For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete deri...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114606
更新日期：2017-11-01 00:00:00
abstract：:Tandemly polymerized regulatory elements, antisense RNA segments or ribozymes are potentially useful in selective gene silencing. However, existing methods of tandemly polymerizing short DNA segments are laborious. We present a procedure that can create cloned arrays of 40-70 monomer units in two steps. We have create...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-11-01 00:00:00
abstract：:A major problem in analyzing forensic casework samples is the presence of genetic material from more than one individual in the material evidence. For instance, in sexual assault cases the evidence (vaginal swabs) usually contains a majority of vaginal epithelial cells and varying amounts of sperm cells from the perpe...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1995-09-01 00:00:00
abstract：:We describe a bead-based, multiplexed, oligonucleotide ligation assay (OLA) performed on the Luminex flow cytometer. Differences between this method and those previously reported include the use of far fewer beads and the use of a universal oligonucleotide for signal detection. These innovations serve to significantly...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112960
更新日期：2008-11-01 00:00:00
abstract：:We describe a method for rapid radioactive labeling of PCR product. The method, employing the Klenow fragment of DNA polymerase I, consumes little product, requires no product purification and takes under 30 minutes. ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-08-01 00:00:00
abstract：:Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of dev...
journal_title：BioTechniques
pub_type：
doi：10.2144/03342st01
更新日期：2003-02-01 00:00:00
abstract：:Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not co...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04371DD02
更新日期：2004-07-01 00:00:00
abstract：:In order to study quantitative gene expression with Northern blots, it is important to have an internal standard that can be used to verify even loading or to correct for uneven loading between lanes. In this study it is shown that two-dimensional quantitation of ethidium bromide-intercalated 28S rRNA fluorescence can...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-12-01 00:00:00
abstract：:A low order neural network-based filter was designed as a rapid screening agent for single-spanning transmembrane regions in an integrated informatics system. A rapid screening algorithm was seen as a compromise between costly structure-specific techniques and simple rules that gave a high false-positive rate for cDNA...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/96216bc01
更新日期：1996-12-01 00:00:00
abstract：:Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3' ends and then PCR amplification of the 3'-end signature tag population. This met...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04362ST02
更新日期：2004-02-01 00:00:00
abstract：:14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially avai...
journal_title：BioTechniques
pub_type：
doi：10.2144/98245st04
更新日期：1998-05-01 00:00:00
abstract：:Here we present a procedure for preparing amino acid mixtures--having both the desired composition and a physiological pH--at high concentrations for cell-free expression systems. Up to 2.1 mg/mL of active protein was synthesized in batch mode reactions with an all Escherichia coli cell-free expression system. Our met...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114249
更新日期：2015-01-01 00:00:00
abstract：:Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before...
journal_title：BioTechniques
pub_type：
doi：10.2144/99266st02
更新日期：1999-06-01 00:00:00
abstract：:A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2) were mixed with trehalose and xanthan at final c...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114207
更新日期：2014-09-01 00:00:00
abstract：:We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 microgr...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/03344pt03
更新日期：2003-04-01 00:00:00
abstract：:A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites. Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1989-03-01 00:00:00
abstract：:A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and e...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1989-11-01 00:00:00
abstract：:Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a l...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1990-11-01 00:00:00
abstract：:Cell culture is a vital component of laboratories throughout the scientific community, yet the absence of standardized protocols and documentation practice challenges laboratory efficiency and scientific reproducibility. We examined the effectiveness of a cloud-based software application, CultureTrax® as a tool for st...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2018-0062
更新日期：2018-11-01 00:00:00
abstract：:Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucum...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112750
更新日期：2008-06-01 00:00:00
abstract：:We have developed an automated purification method for dye-terminator-based DNA sequencing products using a magnetic bead approach. This 384-well protocol generates sequence fragments that are essentially free of template DNA, salt, and excess dye-terminator products. In comparison with traditional ethanol precipitati...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/02326st05
更新日期：2002-06-01 00:00:00
