Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.
Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.
Tarnuzzer
R W
RW
University of Florida, Department of OB/GYN, Gainesville 32610, USA rtarn.obgyn@obgyn.ufl.edu
Macauley
S P
SP
Farmerie
W G
WG
Caballero
S
S
Ghassemifar
M R
MR
Anderson
J T
JT
Robinson
C P
CP
Grant
M B
MB
Humphreys-Beher
M G
MG
Franzen
L
L
Peck
A B
AB
Schultz
G S
GS
D001667:Binding, Competitive / Q000235:genetics; D016207:Cytokines / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D006133:Growth Substances / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification
D016207:Cytokines; D016326:Extracellular Matrix Proteins; D006133:Growth Substances; D012333:RNA, Messenger; D008666:Metalloendopeptidases