RESUMO
Cloning of differentially expressed genes is one of the hottest topics in biology.It is very important in cloning genes correlated with phenotype and diseases at molecular level.Here we utilized a simple and rapid PCR-based protocol to detect and isolate cDNA fragments from differentially expressed genes of IL-6-induccd and IL-6-uninduced U937 cells in tWO easy steps.To generate cDNAs from most mRNAs,the first step was reverse transcription using three fully degenerated 6-mer oligonucleotides as primers.The second step was PGR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences.The PCR amplification was repeated on the same cDNA templates(first step) with different sets of primers.DNA fragments were easily displayed by 2% agarose gel electrophoresis and then the differential recovered fragments were used directly in cloning,sequencing,and RNA reverse Northern blot analysis.In this study,seven differential ESTs are obtained;two Sequences not found in GenBank,are novel ESTs.They were proved to be differentially expressed genes related with IL-6 effect by reverse Northern hybridization.
RESUMO
Cloning of differentially expressed genes is one of the hottest topics in biology.It is very important in cloning genes correlated with phenotype and diseases at molecular level.Here we utilized a simple and rapid PCR-based protocol to detect and isolate cDNA fragments from differentially expressed genes of IL-6-induccd and IL-6-uninduced U937 cells in tWO easy steps.To generate cDNAs from most mRNAs,the first step was reverse transcription using three fully degenerated 6-mer oligonucleotides as primers.The second step was PGR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences.The PCR amplification was repeated on the same cDNA templates(first step) with different sets of primers.DNA fragments were easily displayed by 2% agarose gel electrophoresis and then the differential recovered fragments were used directly in cloning,sequencing,and RNA reverse Northern blot analysis.In this study,seven differential ESTs are obtained;two Sequences not found in GenBank,are novel ESTs.They were proved to be differentially expressed genes related with IL-6 effect by reverse Northern hybridization.