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Cloning differentially expressed mRNAs. 1996

J S Wan, and S J Sharp, and G M Poirier, and P C Wagaman, and J Chambers, and J Pyati, and Y L Hom, and J E Galindo, and A Huvar, and P A Peterson, and M R Jackson, and M G Erlander
R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121, USA.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

UI MeSH Term Description Entries
D007371 Interferon-gamma The major interferon produced by mitogenically or antigenically stimulated LYMPHOCYTES. It is structurally different from TYPE I INTERFERON and its major activity is immunoregulation. It has been implicated in the expression of CLASS II HISTOCOMPATIBILITY ANTIGENS in cells that do not normally produce them, leading to AUTOIMMUNE DISEASES. Interferon Type II,Interferon, Immune,gamma-Interferon,Interferon, gamma,Type II Interferon,Immune Interferon,Interferon, Type II
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D002452 Cell Count The number of CELLS of a specific kind, usually measured per unit volume or area of sample. Cell Density,Cell Number,Cell Counts,Cell Densities,Cell Numbers,Count, Cell,Counts, Cell,Densities, Cell,Density, Cell,Number, Cell,Numbers, Cell
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D006367 HeLa Cells The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays. Cell, HeLa,Cells, HeLa,HeLa Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000998 Antiviral Agents Agents used in the prophylaxis or therapy of VIRUS DISEASES. Some of the ways they may act include preventing viral replication by inhibiting viral DNA polymerase; binding to specific cell-surface receptors and inhibiting viral penetration or uncoating; inhibiting viral protein synthesis; or blocking late stages of virus assembly. Antiviral,Antiviral Agent,Antiviral Drug,Antivirals,Antiviral Drugs,Agent, Antiviral,Agents, Antiviral,Drug, Antiviral,Drugs, Antiviral
D012269 Ribosomal Proteins Proteins found in ribosomes. They are believed to have a catalytic function in reconstituting biologically active ribosomal subunits. Proteins, Ribosomal,Ribosomal Protein,Protein, Ribosomal
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D015152 Blotting, Northern Detection of RNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES. Northern Blotting,Blot, Northern,Northern Blot,Blots, Northern,Blottings, Northern,Northern Blots,Northern Blottings

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