in 5 co2 humidified incubator at 37 degrees c approximately 20 mg of tissue was obtained from each sample for rna extraction using trizol reagent the purification procedure was performed according to the manufacture 39 s protocol the quality of rna samples was determined by electrophoresis on agarose gel and by visualization of the integrity of the 18s and 28s rna bands the concentration purity and amount of total rna were determined by ultraviolet spectrophotometry by these standards all the rna samples used for assay were of high quality and purity abs 260 abs280 gt 1 7 the reverse transcription reaction was performed by incubating a reaction mixture containing 300 ng total rna 6 u03bcl of 5 u00d7 first buffer 250 mm tris hcl ph8 3 375 mm kcl 15 mm mgcl2 20 u03bcm of random haxamer primer 50 u of reverse transcriptase 10 u of rnase inhibitor and 0 5 mm dntp in a total of 30 u03bcl reaction buffer at 42 degrees c for 1 hour primers and probes for each gene were chosen for each primer and probe we conducted a blastn search against the genbank database to confirm the total gene specificity and the absence of dna polymorphism the pcr amplicon regions for nucleophosmin b23 contained all the possible splicing products to ensure the measurement of total mrna expression the primers and probes used for rt pcr are b23.