Cloning differentially expressed mRNAs.
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.
Wan
J S
JS
R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121, USA.
Sharp
S J
SJ
Poirier
G M
GM
Wagaman
P C
PC
Chambers
J
J
Pyati
J
J
Hom
Y L
YL
Galindo
J E
JE
Huvar
A
A
Peterson
P A
PA
Jackson
M R
MR
Erlander
M G
MG
D000998:Antiviral Agents / Q000494:pharmacology; D015152:Blotting, Northern; D002452:Cell Count; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D009693:Nucleic Acid Hybridization / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics
D000998:Antiviral Agents; D018076:DNA, Complementary; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D007371:Interferon-gamma