Construction And Analization Of Serial Analysis Of Gene Expression Library Of Peripheral Blood Mononuclear Cells In Patients With Systemic Lupus Erythematosus

Partâ… Construction of Serial Analysis of Gene Expression Library of Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus ErythematosusObjective:To construct serial analysis of gene expression(SAGE) library of peripheral blood mononuclear cells(PBMCs)in Patients with systemic lupus erythematosus(SLE)Methods:PBMCs were exstracted from five milliliter of peripheral blood of three patients with family SLE respectively as original material.SAGE library was constructed by means of SAGE technique.Three modifications were applied to SAGE technique compared to the original technique:1)mRNA was exstracted directly from PBMCs by megnetic beads with oligo(dT)25 cDNA synthesis and digestion with Nlaâ…¢were performed in the same tube which avoided DNA loss due to extraction with phenol and chloroform and precipitation with ethanol in the original SAGE technique.2)A limited number of additional PCR cycles were performed to obtain suffient 100bp ditag. 3) pZErO?-1 vector was used to improve the clone efficiency.Two hundred and thirty-seven colonies were randomly picked ,colony PCR was applied to know weather the recombinant DNA were transducted into the bacteria and the length of the insert DNA.Sequencing were performed to some positive colonies to verify that the arrangement of the bases of insert DNA suit to the arrangement of SAGE tags.Results:1600,000 PBMCs were extracted from peripheral blood of three patients with family SLE. SAGE library of PBMCs in patients with SLE was set up and six hundred colonies were obtained.Two hundred and thirty seven colonies were ramdomly picked to be analyzed by colony PCR of which two hundred and sixteen colonies were confirmed that cDNA with length more than 250 bp were inserted,almost ninty-one percent of positive clonies contained inserts.Each insert DNA contained about ten to sixties tags in which ten to forty tags were commonly seen.Subsequent sequencing verified that each insert DNA contained several hundred bases which was characterized by that about twenty based were comparted by"CATG"which was in line with the arrangement of tags of SAGE.Conclusion:Three modifications were applied to original SAGE technique so that it can be applied to minite quantities of tissues or cells.SAGE library of PBMCs in patients with SLE was successfully constructed which helped to understand the gene expression profile of PBMCs in patient with SLE and laid a foundation to screen differentially expressed genes in patients with SLE.Partâ…¡A Pilot Study of Serial Analysis of Gene Expression of Peripheral Blood Mononuclear Cells in Patients with SLEObjective:To outline the gene expression patterns of peripheral blood mononuclear cells in patients with SLE.Methods:Following construction of SAGE library of PBMCs in patients with family SLE,ninty-eight colonies were randomly picked to be sequenced.Tag sequences were analized by SAGE 2000 V 4.5 software.Human SAGE tag database was downloaded from web of http://www.ncbi.nlm.nih.gov/SAGE map,tag sequences were compared to the database to obtain the corresponding genes expressed in PBMCs in patients with SLE.Top thirty tags in library were submited to the web of http://www.ncbi.nlm.nih.gov/SAGE to obtain the corresponding genes and their functions.Results:A total of 1814 unique SAGE tags were identified from which 1286 tags were distinct .Among the 1286 identified unique tags, 86.8% were single copies, 13% were between 2 and 19 copies, and only 0.2% of tags were more than 20 copies. 68.4% of the tags matched known expressed sequences, 41.1% of which were matched to more than one known expressed sequences;31.6% of the tags had no match and represent potential novel genes.Top thirty genes in the library were ribosomal genes and genes involved in basic metabolism.Conclusion:The present study draws an outline map of gene expression patterns of PBMCs in patients with SLE.More tags obtained from further sequencing would help to obtain complete gene expression profile and comparison the SAGE database between normal individuals and patients with SLE will benefit us to screen the differentially expressed genes in SLE after SAGE library of PBMCs in normal control is constructed.