-
Proceedings of the National Academy of... Dec 2002The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF...
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).
Topics: Algorithms; Animals; DNA, Complementary; Gene Library; Humans; Mice; Open Reading Frames; Sequence Analysis, DNA
PubMed: 12477932
DOI: 10.1073/pnas.242603899 -
Genome Biology Jun 2002Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for...
BACKGROUND
Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.
RESULTS
We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.
CONCLUSIONS
The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
Topics: Algorithms; DNA, Complementary; Female; Gene Expression Profiling; Humans; RNA; Reference Standards; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tumor Cells, Cultured
PubMed: 12184808
DOI: 10.1186/gb-2002-3-7-research0034 -
BMC Biology Sep 2022Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA....
BACKGROUND
Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult.
RESULTS
Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods.
CONCLUSIONS
SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.
Topics: DNA; DNA, Complementary; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA; Sequence Analysis, RNA; Single-Cell Analysis; Transcription Factors
PubMed: 36175891
DOI: 10.1186/s12915-022-01416-x -
RNA (New York, N.Y.) Jul 2023RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the... (Review)
Review
RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.
Topics: Reverse Transcription; DNA, Complementary; Artifacts; RNA; Sequence Analysis, RNA; Bias
PubMed: 36990512
DOI: 10.1261/rna.079623.123 -
Current Protocols in Molecular Biology Jan 2019This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing...
This unit describes a reverse transcription-quantitative PCR (RT-qPCR)-based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; DNA, Complementary; Gene Expression Profiling; Gene Expression Regulation; Humans; Protein Biosynthesis; Proteins; RNA; Real-Time Polymerase Chain Reaction; Reverse Transcription; Transcription, Genetic
PubMed: 30346115
DOI: 10.1002/cpmb.71 -
Wiley Interdisciplinary Reviews. RNA 2011Methods for in-depth genome-wide characterization of transcriptomes and quantification of transcript levels using various microarray and next-generation sequencing... (Review)
Review
Methods for in-depth genome-wide characterization of transcriptomes and quantification of transcript levels using various microarray and next-generation sequencing technologies have emerged as valuable tools for understanding cellular physiology and human disease biology and have begun to be utilized in various clinical diagnostic applications. Current methods, however, typically require RNA to be converted to complementary DNA prior to measurements. This step has been shown to introduce many biases and artifacts. In order to best characterize the 'true' transcriptome, the single-molecule direct RNA sequencing (DRS) technology was developed. This review focuses on the underlying principles behind the DRS, sample preparation steps, and the current and novel avenues of research and applications DRS offers.
Topics: DNA, Complementary; Gene Expression Profiling; Genome, Human; Humans; Oligonucleotide Array Sequence Analysis; Optical Devices; Sequence Analysis, RNA
PubMed: 21957044
DOI: 10.1002/wrna.84 -
Chembiochem : a European Journal of... Apr 2021RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA... (Review)
Review
RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA structure and are now considered invaluable resources in the goal of relating structure to function. Recently, the power of deep sequencing and careful chemical probe design have merged, permitting researchers to obtain a holistic understanding of how RNA structure can be utilized to control RNA biology transcriptome-wide. Within this review, we outline the recent advancements in chemical probe design for interrogating RNA structures inside cells and discuss the recent advances in our understanding of RNA biology through the lens of chemical probing.
Topics: DNA Adducts; DNA, Complementary; Molecular Probes; Nucleic Acid Conformation; RNA; RNA, Messenger; Transcriptome
PubMed: 32737940
DOI: 10.1002/cbic.202000340 -
Biochemistry Sep 2017DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the... (Comparative Study)
Comparative Study
DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.
Topics: Base Pairing; DNA; DNA, Complementary; Fluorescence Resonance Energy Transfer; GC Rich Sequence; Models, Molecular; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Single Molecule Imaging; Spectrometry, Fluorescence
PubMed: 28820590
DOI: 10.1021/acs.biochem.7b00677 -
Briefings in Functional Genomics &... Nov 2009Non-coding RNA (ncRNA) transcripts are RNA molecules that do not code for proteins, but elicit function by other mechanisms. The vast majority of RNA produced in a cell... (Review)
Review
Non-coding RNA (ncRNA) transcripts are RNA molecules that do not code for proteins, but elicit function by other mechanisms. The vast majority of RNA produced in a cell is non-coding ribosomal RNA, produced from relatively few loci, however more recently complementary DNA (cDNA) cloning, tag sequencing, and genome tiling array studies suggest that ncRNAs also account for the majority of RNA species produced by a cell. ncRNA based regulation has been referred to as a 'hidden layer' of signals or 'dark matter' that control gene expression in cellular processes by poorly described mechanisms. These terms have appeared as ncRNAs until recently have been ignored by expression profiling and cDNA annotation projects and their mode of action is diverse (e.g. influencing chromatin structure and epigenetics, translational silencing, transcriptional silencing). Here, we highlight recent functional genomics strategies toward identifying and assigning function to ncRNA transcription.
Topics: Animals; Computational Biology; DNA, Complementary; Genomics; Humans; RNA, Untranslated; Transcription, Genetic
PubMed: 19833699
DOI: 10.1093/bfgp/elp041 -
BioTechniques Mar 2012
Topics: Animals; DNA; DNA, Complementary; Dogs; Nucleic Acid Amplification Techniques; RNA; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease H; Sensitivity and Specificity; Skin
PubMed: 22401544
DOI: 10.2144/000113817