Simultaneous Quantitation of Cytokine mRNAs by Reverse Transcription-Polymerase Chain Reaction Using Multiple Internal Standard cRNAs

Abstract

Cytokines produced in abnormal amounts or patterns contribute to many immunologically mediated human diseases. We describe a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure interleukin (IL)1-2, IL-4, and interferon-gamma (IFN-gamma) mRNAs within the sample. Internal standard cRNAs and native cytokine mRNAs are reverse transcribed and then amplified by PCR in the same reaction tubes to control for tube-to-tube variability in these reactions. In contrast to systems that use a single multigene internal standard cRNA, this method uses separate internal standard cRNAs for IL-2, IL-4, and IFN-gamma, allowing independent dosing of the internal standards, which reduces the number of tubes processed and the amount of starting mRNA required. Internal standards are produced from cytokine cDNAs by the insertion of short segments of DNA. The same oligonucleotide primers are used to amplify internal standard and native cytokine cDNAs. Each internal standard cDNA and its matching native cytokine cDNA are amplified with equal efficiency. The RT-PCR products of the internal standards and native cytokines are distinguished by size. This technique can detect a twofold difference in mRNA levels. Examples of using this technique to measure cytokine mRNAs in peripheral blood mononuclear cells and in bronchoalveolar lavage cells are given.

Overexpression of the insulin‐like growth factor I receptor in human colon carcinomas

“…The MIMIC PCR technique utilizes an exogenous internal standard (MIMIC) that competes for the same primers as the target IGF‐IR or IGF‐II DNA. With knowledge of the amount of MIMIC DNA added in serial dilutions to the amplification reactions, the amount of the target template can be determined, and thus the initial IGF‐IR or IGF‐II mRNA concentration 29, 30. The competitive internal standard, which contains the identical primer binding sites used to amplify the IGF‐IR or IGF‐II DNA, was generated by amplifying a Bam H1/ Eco R1 fragment of v‐erB with two composite primers.…”

Overexpression of the insulin-like growth factor I receptor in human pheochromocytomas

“…Subsequently, a cycle number that was in the middle of the linear amplification range (21-27 cycles) was chosen. In the system used, the efficiencies of amplification of target cDNA and competitive internal standard DNA were equal (Gilliland et al 1990, Alms et al 1996, Becker-Andre et al 1989, Kutoh et al 1998.…”

“…After RT-PCR, the ratios of MIMIC to target band intensity were determined, and the concentration of a 1:1 MIMIC/target ratio was calculated as described (Kutoh et al 1998). For each sample, an initial estimate of IGF-IR mRNA was performed with a single dose of internal standard DNA, followed by a narrow titration of internal standards around this estimated value, according to the method of Alms et al (1996). Results were expressed as number of molecules per µg total RNA.…”

The Inherent Quantitative Capacity of the Reverse Transcription-Polymerase Chain Reaction

“…RNA solutions were brought to equal concentrations, and HSV-1-infected Vero cell RNA was serially diluted using uninfected Vero cell RNA as a diluent. Firststrand cDNA was synthesized from total RNA using an oligo(dT 15 ) primer and AMV reverse transcriptase (Promega Corp., Madison, WI) in a 20 l volume containing 100 ng RNA per l. Reactions were incubated at 42°C for 45 min, followed by 5 min at 99°C to inactivate reverse transcriptase.…”

“…Because PCR is an exponential process, a primary source of concern is that large tube-totube variability in product yield will result from initially minor tube-to-tube differences (1-3, 8 -15). To overcome this obstacle, competitive PCR techniques have been developed in which an internal control (i.e., competitor DNA template) is included in each reaction and the target PCR product yield is compared to the competitor PCR product yield (2,3,9,10,15,16). Because the ratio of target to competitor PCR products is independent of amplification efficiency, tube-to-tube variability does not confound estimates of target abundance (4).…”