1995
DOI: 10.1155/s0962935195000329
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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 includ… Show more

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Cited by 6 publications
(6 citation statements)
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“…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.…”
Section: Pcrmentioning
confidence: 99%
“…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.…”
Section: Pcrmentioning
confidence: 99%
“…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.…”
Section: Lymphocyte Proliferation Assaymentioning
confidence: 99%
“…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.…”
Section: Analysis Of Il-4 and Ifn-g Mrna Expression And Production Inmentioning
confidence: 99%
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“…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.…”
Section: Rna Isolation and Northern Blot Analysismentioning
confidence: 99%