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Cloning Of Preeclampsia Related Gene By Suppression Subtractive Hybridization

Posted on:2004-05-11Degree:MasterType:Thesis
Country:ChinaCandidate:K R ZhangFull Text:PDF
GTID:2144360092999232Subject:Gynecology
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Objective: To construct a cDNA subtractive library and to screen human preeclampsia related gene with suppression subtractive hybridizarion technipue. The library contains highly expressed or new cDNA in preeclampsia comparing with normal pregnancy.Methods: (1) RNA were abstracted from cesarean delivered placenta of preeclampsia and normal pregnancy, then poly(A)+ RNA was purified. To estimate the concentration and purification of RNA,the ratio of 260/280nm was calculated. At same time, the RNA'S integrality was examined by electrophoresing on a formaldehyde denaturing agarose/EtBr gel. (2) cDNA were synthesized with AMV reverse transcriptase. After enzyme restriction, cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively. (3) After cDNA from preeclamtic placenta were hybridized with normal pregnancy cDNA twice and underwent twice nested PCR. cDNA was then ligated with arms of T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out through transfecting E.coli strain JM 109. White and blue clonies were screened in Jm 109 on LB/X-Gal/IPTG/Amp plates. (4) The subtracted cDNA library was hybridized with reversly-subtracted probes, then positive clones were selected. The gene sequences were analyzed. (5) In order to observe the expression of the differentially expressed cDNA in placental samples, dot bloting hybridization was performed between differentially expressed cDNA and mRNA abstracted from placenta.The statictic significance was test by X2-test.Results: (1) RNA Samples' integrality The RNA and mRNA samples were electrophoresised on a 1% formaldehyde denaturing agarose/EtBr gel. The vatio of 28s/18s band density equaled to 1.5, and mRNA were shownas smear (2) Analysis of Rsal-digested cDNA The undigested and Rsal-digested ds cDNA was electrophoresised on a 1% agarose/EtBr gel.Bright bands showed that the average size of Rsal- digested cDNA was smaller(0.1-2kb compare to 0.5-10 Kb)than that of undigested. (3) Analysis of ligation After 35 PCR cycles,the ligated cDNA was electrophoresised on a 1% agarose/EtBr gel. It showed that the PCR product amplified using one gene-specific primer and PCR Primer 1 was nearly as the same in intensity as the PCR product amplified using two gene-specificfprimers. (4) PCR anslysis of subtractive efficiency (5) Cloning the PCR products and differential screening by blue/white blot After cloned into the PT-Adv, the PCR product were introduced into the JM109 coli competent cells.Then the transformation was spread onto the LB/X-GaL/PTG plates, and the 60 white clone were selected (Abtain the cDNA Library). (6) Verification of the white clones by dot bloting hybridization The 60 white clones were hybridizated with substracted cDNA from preeclantic placenta. 26 white clones were found faks positive. (7) Blast analysis of the cDNA sequences of positive white clones The cDNA sequences of positive white clones were examed by gene chip and were analyze by blast program. 4 highly expressed genes were found in pre-aclamptic placenta ,those included decorin mRNA, insulin-like growth factor binding protein mRNA and two mitochondrion genes. (8) verification of the gene ecpression in more placental samples. DCN and IGFBP-1 Cdna were selected as the probes and were hybridizated with Mrna samples from including 32 pre-eclamptic placentas and 32 normal placentas) .Statistical analysis was performed by using the X2- test. P<0.01 was considered statistically significant. Conclusions Our studies showed that, compared with placenta of normal pregnancy, transparently highly expression of decorin, insulin-like growth factor binding protein -1 and mitochondrion genes were found in placenta of preeclampsia, which might be involved in the pathogenesis of preeclampsia.
Keywords/Search Tags:preeclampsia, hybridization, decorin, insulin-like growth factor binding protein-1, mitochondrion, subtractive hybridization, clone, placenta
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