Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection.


:Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce 'primer dimer' formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.


Nucleic Acids Res


Nucleic acids research


Stanley KK,Szewczuk E





Has Abstract


2005-11-24 00:00:00














  • Structure-function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop-loop interaction motif conserved in most natural hammerheads.

    abstract::Loop-loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (-) hammerheads of ...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Dufour D,de la Peña M,Gago S,Flores R,Gallego J

    更新日期:2009-02-01 00:00:00

  • The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle.

    abstract::Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes. In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair. To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photol...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Al-Moghrabi NM,Al-Sharif IS,Aboussekhra A

    更新日期:2001-05-15 00:00:00

  • Physical map of Neurospora crassa mitochondrial DNA and its transcription unit for ribosomal RNA.

    abstract::A circular denaturation and restriction map of mitochondrial DNA from Neurospora crassa is presented. The map shows the position of all twelve fragments produced by restriction endonuclease Eco R I and the position of the largest Hin III fragment along the previously established map of AT-rich sequences. The two wild ...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Bernard U,Goldthwaite C,Küntzel H

    更新日期:1976-11-01 00:00:00

  • Protection of (dA.dT) cluster regions in the DNAase I cleavage of DNA by specific interaction with netropsin.

    abstract::The specific DNA binding ligand netropsin selectively blocks dA-dT base pairs in clusters containing two or more consecutive thymine residues at the dNAase I cleavage sites of DNA. Using CD and UV absorption measurements it is shown, that at various ratios of netropsin to nucleotide concentrations and even at satuatio...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Zimmer C,Luck G,Nüske R

    更新日期:1980-07-11 00:00:00

  • Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres.

    abstract::Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Rao KV,Stevens PW,Hall JG,Lyamichev V,Neri BP,Kelso DM

    更新日期:2003-06-01 00:00:00

  • Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds.

    abstract::Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-solu...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Grigg GW,Gero AM,Sasse WH,Sleigh MJ

    更新日期:1984-12-11 00:00:00

  • Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates.

    abstract::The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to ...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Lagerbäck P,Andersson E,Malmberg C,Carlson K

    更新日期:2009-10-01 00:00:00

  • Methyl transferases induced during chemical adaptation of M. luteus.

    abstract::Three peaks of methyltransferase activity specific for MNNG alkylated DNA have been identified from extracts of chemically adapted M. luteus. They are designated as TI to TIII in order to their elution from a Sephadex G-75 column. The first one of these peaks has been purified to homogeneity. TI, is an inducible, unus...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Riazuddin S,Athar A,Sohail A

    更新日期:1987-11-25 00:00:00

  • Aspects of specific protein-DNA interaction; multi-mode binding of the oligopeptide antibiotic netropsin to (A.T)-rich DNA segments.

    abstract::By means of titration viscometry a number of distinct modes could be resolved for the interaction between the antibiotic netropsin and DNA species of 50, 58, and 69 mole + (A+T) below r = 0.04 netropsin molecules bound per DNA phosphate group. The number of corresponding binding sites increases with a high power of th...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Reinert KE,Stutter E,Schweiss H

    更新日期:1979-11-10 00:00:00

  • Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.

    abstract::We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Charnay P,Perricaudet M,Galibert F,Tiollais P

    更新日期:1978-12-01 00:00:00

  • Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.

    abstract::The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replicat...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Fisher RJ,Fivash MJ,Stephen AG,Hagan NA,Shenoy SR,Medaglia MV,Smith LR,Worthy KM,Simpson JT,Shoemaker R,McNitt KL,Johnson DG,Hixson CV,Gorelick RJ,Fabris D,Henderson LE,Rein A

    更新日期:2006-01-24 00:00:00

  • Summaries of Affymetrix GeneChip probe level data.

    abstract::High density oligonucleotide array technology is widely used in many areas of biomedical research for quantitative and highly parallel measurements of gene expression. Affymetrix GeneChip arrays are the most popular. In this technology each gene is typically represented by a set of 11-20 pairs of probes. In order to o...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Irizarry RA,Bolstad BM,Collin F,Cope LM,Hobbs B,Speed TP

    更新日期:2003-02-15 00:00:00

  • The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium.

    abstract::DNA and RNA guanine-quadruplexes (G4s) are stabilized by several cations, in particular by potassium and sodium ions. Generally, potassium stabilizes guanine-quartet assemblies to a larger extent than sodium; in this article we report about a higher-order G4 structure more stable in sodium than in potassium. Repeats o...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Saintomé C,Amrane S,Mergny JL,Alberti P

    更新日期:2016-04-07 00:00:00

  • Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex.

    abstract::Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine-acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N',N'-tetraacetate (...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Yamamoto Y,Tsuboi W,Komiyama M

    更新日期:2003-08-01 00:00:00

  • DRB4 dsRBD1 drives dsRNA recognition in Arabidopsis thaliana tasi/siRNA pathway.

    abstract::In Arabidopsis thaliana, endogenous trans-acting and exogenous siRNA pathways are initiated by the interaction of DRB4 with trigger dsRNA. Further, DCL4:DRB4 complex cleaves the dsRNA into 21 bp siRNA. Understanding molecular determinants and mechanistic details of dsRNA recognition by DRB4 is vital for inducing long-...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Chiliveri SC,Aute R,Rai U,Deshmukh MV

    更新日期:2017-08-21 00:00:00

  • Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease.

    abstract::Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1-7 nt) and sequence composition were formed in a 3' terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulg...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Kuznetsova IL,Zenkova MA,Gross HJ,Vlassov VV

    更新日期:2005-02-24 00:00:00

  • LOCnet and LOCtarget: sub-cellular localization for structural genomics targets.

    abstract::LOCtarget is a web server and database that predicts and annotates sub-cellular localization for structural genomics targets; LOCnet is one of the methods used in LOCtarget that can predict sub-cellular localization for all eukaryotic and prokaryotic proteins. Targets are taken from the central registration database f...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Nair R,Rost B

    更新日期:2004-07-01 00:00:00

  • The mapping of nucleosomes and regulatory protein binding sites at the Saccharomyces cerevisiae MFA2 gene: a high resolution approach.

    abstract::We have developed an end-labelling approach to map the positions of nucleosomes and protein binding sites at nucleotide resolution by footprinting micrococcal nuclease (MNase)-sensitive sites. Using this approach we determined that the MFA2 gene and its upstream control regions have four positioned nucleosomes when tr...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Teng Y,Yu S,Waters R

    更新日期:2001-07-01 00:00:00

  • Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications.

    abstract::Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analy...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Rutledge RG

    更新日期:2004-12-15 00:00:00

  • CAMP: Collection of sequences and structures of antimicrobial peptides.

    abstract::Antimicrobial peptides (AMPs) are gaining importance as anti-infective agents. Here we describe the updated Collection of Antimicrobial Peptide (CAMP) database, available online at The 3D structures of peptides are known to influence antimicrobial activity. Although there exists datab...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Waghu FH,Gopi L,Barai RS,Ramteke P,Nizami B,Idicula-Thomas S

    更新日期:2014-01-01 00:00:00

  • Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing.

    abstract::The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of mus...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Banerjee A,Vest KE,Pavlath GK,Corbett AH

    更新日期:2017-10-13 00:00:00

  • The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species.

    abstract::In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In tRNA(cmnm...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Moukadiri I,Garzón MJ,Björk GR,Armengod ME

    更新日期:2014-02-01 00:00:00

  • Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells.

    abstract::DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers t...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Kong Q,Maizels N

    更新日期:2001-03-15 00:00:00

  • Investigating the role of the Est3 protein in yeast telomere replication.

    abstract::The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Lee J,Mandell EK,Rao T,Wuttke DS,Lundblad V

    更新日期:2010-04-01 00:00:00

  • The dynamics of chromatin carcinogen interactions in the human cell.

    abstract::Human lung epithelioid cells were treated with Benzo (a) pyrene diol epoxide (anti) in order to establish the binding and removal of covalent adducts in chromosomal components. Isolating two different classes of mononucleosomes, it was found that their DNA contained different concentrations of B(a)PDE-DNA adducts, whi...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Kootstra A

    更新日期:1986-12-22 00:00:00

  • MODPROPEP: a program for knowledge-based modeling of protein-peptide complexes.

    abstract::MODPROPEP is a web server for knowledge-based modeling of protein-peptide complexes, specifically peptides in complex with major histocompatibility complex (MHC) proteins and kinases. The available crystal structures of protein-peptide complexes in PDB are used as templates for modeling peptides of desired sequence in...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Kumar N,Mohanty D

    更新日期:2007-07-01 00:00:00

  • In vivo cleavage rules and target repertoire of RNase III in Escherichia coli.

    abstract::Bacterial RNase III plays important roles in the processing and degradation of RNA transcripts. A major goal is to identify the cleavage targets of this endoribonuclease at a transcriptome-wide scale and delineate its in vivo cleavage rules. Here we applied to Escherichia coli grown to either exponential or stationary...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Altuvia Y,Bar A,Reiss N,Karavani E,Argaman L,Margalit H

    更新日期:2018-11-02 00:00:00

  • Specific reverse transcriptase slippage at the HIV ribosomal frameshift sequence: potential implications for modulation of GagPol synthesis.

    abstract::Synthesis of HIV GagPol involves a proportion of ribosomes translating a U6A shift site at the distal end of the gag gene performing a programmed -1 ribosomal frameshift event to enter the overlapping pol gene. In vitro studies here show that at the same shift motif HIV reverse transcriptase generates -1 and +1 indels...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Penno C,Kumari R,Baranov PV,van Sinderen D,Atkins JF

    更新日期:2017-09-29 00:00:00

  • Summarizing and correcting the GC content bias in high-throughput sequencing.

    abstract::GC content bias describes the dependence between fragment count (read coverage) and GC content found in Illumina sequencing data. This bias can dominate the signal of interest for analyses that focus on measuring fragment abundance within a genome, such as copy number estimation (DNA-seq). The bias is not consistent b...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Benjamini Y,Speed TP

    更新日期:2012-05-01 00:00:00

  • AH-DB: collecting protein structure pairs before and after binding.

    abstract::This work presents the Apo-Holo DataBase (AH-DB, and, which provides corresponding pairs of protein structures before and after binding. Conformational transitions are commonly observed in various protein interactions that are involved in important biological f...

    journal_title:Nucleic acids research

    pub_type: 杂志文章


    authors: Chang DT,Yao TJ,Fan CY,Chiang CY,Bai YH

    更新日期:2012-01-01 00:00:00