Abstract
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.
Highlights
We describe a simplified and reliable polymerase has been lowered1000-fold [2,3,4,5,6,7]
With the polymerase chain reaction (PCR),’ the detection threshold mRNA assay, we have explored the possibility of exploiting the technique during both the exponential and nonexponential phases of amplification
We have examined whether the amplification of the cellular template and the internal standards occurs in a parallel fashion throughout the PCR
Summary
We describe a simplified and reliable polymerase has been lowered1000-fold [2,3,4,5,6,7]. Multispecific by up to 2 orders of magnitude This finding means synthetic standards having the same primer hybridization that preliminary experiments designed to determine sequences as those on the RNAs to be assayed have been either the lateexponential region or the amplification reported [13, 15]. This restriction has meant that preliminary calibration studies for each individual template have had to be carried out, making the approach time-consuming and expensive, where extensive surveys are concerned To overcome this limitation and simplify the Antigen activation of lymphocytes results inthe production of a wide range of cytokines acting on both cellular and humoral responses. The abbreviations used are: PCR, polymerase chain reaction; the complementary 5’ and 3’ primer sequences of 15 target genes, we report here that the amplifications of the internal standard and the cellular template are parallel up to the plateau phase. We describe an optimized separation procedure which leads to a precise assay of the amplicons
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