Analysis of cyt0kine gene expression in stimulated T cells of small children by semi‐quantitative PCR

Abstract

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-γ. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0–4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-γ; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.

Somatostatin receptor subtype expression in cells of the rat immune system during adjuvant arthritis

“…Briefly, 2-fold serial dilutions of the cDNA preparations were normalised to give approximately equal -actin signals, before performing PCR for the sst subtypes. The linearity of the dilution series was assessed by scanning the photographs of the ethidium bromide-stained gels and determining the intensity of the bands (Koning et al 1995). For the most accurate quantification, care was taken to include a dilution of which the -actin signal was no longer visible when analysed on ethidium bromide-stained gel.…”

CD4+ cells proliferate after peanut‐extract‐specific and CD8+ cells proliferate after polyclonal stimulation of PBMC of children with atopic dermatitis

“…Five millilitres of heparinized venous blood was collected and PBMC were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia) [32]. PBMC were washed two times and resuspended in RPMI-1640 supplemented with 2 mm L-glutamine, 100 IU/mL penicillin, 50 mg/mL streptomycin, 1 mm pyruvate and 10% heat-inactivated human serum pooled from healthy donors.…”

“…After harvesting the cells, supernatants were stored at -80 ЊC and RNA was isolated from the cells by the RNAzol B method [36] (Cinna-Biotecx Laboratories Inc., Houston, TX, USA). As described previously [32], 1 mg RNA was used for cDNA synthesis and amplification by semi-quantitative RT-PCR using primers specific for either the housekeeping gene HPRT (hypoxanthine phosphoribosyl transferase), IL-4 or IFNg [32]. After the PCR amplification, the five samples, taken after five different cycle numbers, were loaded on a 1.2% agarose gel (Sea Kem Leagarose, FMC Bio Products, Rockland, ME, USA), stained with 75 mg/500 mL ethidium bromide (Boehringer Mannheim, Germany); 0.5 ml PhiX174 (HaeIII digest, 0.5 mg/mL, New England Biolabs, Beverly, MA, USA) was used as marker.…”

“…A life-size photo was taken and used for scanning. Collected scan values were analysed as follows: the mean scan values were calculated at two different cycle numbers (30)(31)(32)(33)(34)(35) cycles for HPRT and IFNg, 35-40 cycles for IL-4), in the linear phase of the PCR reaction. In order to correct for interexperimental variation a batch of cDNA of B21 (a Th 0 clone, Paliard [37]) was used for standardization of HPRT, IFNg and IL-4 expression.…”

Modulation of glucocorticoid receptor expression in human bronchial epithelial cell lines by IL-1 beta, TNF-alpha and LPS

“…Computer software described by KONING et al [19] was used to analyze the intensity of the bands. Values were expressed as the ratio of GR mRNA intensity to GAPDH mRNA intensity.…”