Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.
We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.
Bouaboula
M
M
Sanofi Recherche, Montpellier, France.
Legoux
P
P
Pességué
B
B
Delpech
B
B
Dumont
X
X
Piechaczyk
M
M
Casellas
P
P
Shire
D
D
D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004277:DNA, Single-Stranded; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015375:Receptors, Interleukin-2 / Q000235:genetics; D017186:Titrimetry; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics
D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D015375:Receptors, Interleukin-2; D014409:Tumor Necrosis Factor-alpha; D001613:beta 2-Microglobulin; D015847:Interleukin-4; D007371:Interferon-gamma; D016572:Cyclosporine