Quantification of Cytokine Gene Expression Using an Economical Real‐Time Polymerase Chain Reaction Method Based on SYBR^® Green I

Abstract

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR 1 Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR 1 Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

The effect of an anti-inflammatory pentapeptide produced by Entamoeba histolytica on gene expression in the U-937 monocytic cell line

“…The constitutive gene expression of HPRT was similar between the control and stimulated samples (Ct values 18 29.76 for RPMI, PMA, PMA+MLIF, and MLIF respectively). The specifi c HPRT Tm peak (79 °C) was observed in all samples.…”

“…CRL-1593.2) (a model used for functions of macrophages biological), in response to the treatment with the anti-infl ammatory MLIF pentapeptide using microarrays for gene expression and complemented by real-time RT-PCR and protein determination [16][17][18].…”

MAPK-activated Protein Kinase 2 Differentially Regulates Plasmodium falciparum Glycosylphosphatidylinositol-induced Production of Tumor Necrosis Factor-α and Interleukin-12 in Macrophages

“…Aliquots of cDNA samples were analyzed for TNF-␣, IL-12p40, and ␤-actin (endogenous control) genes by PCR. The primers used were as follows: TNF-␣, forward 5Ј-CGG TGC CTA TGT CTC AGC CT-3Ј, reverse 5Ј-TTG GGC AGA TTG ACC TCA GC-3Ј; IL-12p40 (25), forward 5Ј-CAG AAG CTA ACC ATC TCC TGG TTT G-3Ј, Reverse 5Ј-CCG GAG TAA TTT GGT GCT TCA CAC-3Ј; and ␤-actin, forward 5Ј-ACC CTA AGG CCA ACC GTG AA-3Ј, reverse 5Ј-CCG CTC GTT GCC AAT AGT GA-3Ј. The conditions used for PCR were as follows: for TNF-␣ and ␤-actin, 30 cycles of 94°C denaturation for 30 s, 50°C annealing for 30 s, and 72°C extension for 60 s; for IL-12p40, 40 cycles of 94°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 60 s.…”

The pro-opiomelanocortin genes in rainbow trout (Oncorhynchus mykiss): duplications, splice variants, and differential expression

“…Reactions were carried out in triplicate and each consisted of 0·25 µl of the first-strand cDNA reaction, 0·5 µM each primer, and 1 SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a volume of 15 µl. Thermal cycling profile was 50 C for 2 min, 95 C for 10 min and 40 cycles of 95 C for 30 s, 60 C for 20 s, 72 C for 30 s, and 79·5 C for 10 s. Data collection took place between the 79·5 C step and the denaturation step to avoid measurement of primer dimer artifacts (Ramos-Payan et al 2003). A final dissociation step was performed to assess the specificity of the reaction.…”