| Glioblastoma Multiforme is one of the most common primary tumors in the central nervous system in adult that is highly malignant and difficult to treat, whose prognosis is poor. Right now, highly malignant and invasive are the two big problems with the therapy of glioblastomas .With the development of fundamental investigation, more and more evidences confirm that multiple gene cluster alterations under the influcence of all kinds of factors may contribute to the initiation and progression of gliomas .But at present the most important genetic alterations which are associated with glioblastoma proliferation and invation have not been found. Suppression subtractive hybridization (SSH) is a newly emerged powerful molecular genetic techniques for the generation of subtracted cDNA libraries. Because of its high sensitivity ,efficient and specificity ,it has been widely used in generation of subtracted cDNA libraries by many scholars.From our experiment ,we want to use technique of suppression subtractive hybridization to find out the differential expression genes between glioblastomas and astrocytomas grade I , to screen out genes associate with glioblastoma proliferation and invasion ,and then we can use many other powerful molecular genetic techniques to analysis their functions .1 Clone the difference expression genesObjective Using suppression subtractive hybridization to isolate subtracted cDNA libraries of differentially expressed between human glioblastomas and astrocytomas grade I, and clone gene cluster associate with glioblastoma proliferation and invasion. Method Total RNA are extracted from the collection of glioblastomas and astrocytomas that were resected in Neurosurgery department of ChangZheng Hospital. From the total RNA ,we pure mRNA with the NucleoSpin mRNA Purification Kits.After total and poly A+ isolation ,examine the RNA's integrity by electrophoresing samples on a formaldehyde denaturing agarose/EtBr gel.Total RNA exhibits two typically bright bands,which correspond to ribosomal 28S and 18S RNArespectively, with a ratio of intensities of about 2:1.Poly A+appears as a smear from 0.5-12kb .After setting tester and driver groups,using AMV reverse transcriptase and cDNA synthesis primer synthesis cDNA in order of first-strand and second-strand. The tester and driver cDNA are digested with Rsa I, a four -base-cutting restriction enzyme that yields blunt ends. Electrophorese undigested and Rasl-digested cDNA on a 1% agarose/EtBr gel side-by-side ,cDNA derived from polyA+RNA appears as a smear from 0.5-10kb,bright bands correspond to abundant mRNA .After Rsal digestion, the average cDNA size is smaller (0.5-1.0kb),demonstrate that the digestion process is succeed.The tester cDNA is then subdivided into two portions, and by T4 DNA Ligase, each portion is ligated with a different cDNA adaptor that has stretches of identical sequence to allow annealing of the PCR primer once the recessed ends have been filled in . We perform the following PCR experiment to verify how many cDNA have adaptors on both ends .The experiment is designed to amplify fragments that span the adaptor/cDNA junctions of Tester-1 and Tester-2.G3PDH gene is the special fragment. After 28 cycles, we analyze the product by gel electrophoresis, the PCR product using G3PDH 3' primer and PCR primer 1 is about the same intensity as the PCR product amplified using G3PDH 3' 5' primers. Results of the ligation efficiency are satisfied.An excess of astrocytomas grade I driver cDNA is added to each glioblastomas tester cDNA to perform the first forword-hybridization ,and the samples are heat denatured and allowed to anneal.The two samples from the first hybridization are mixed together ,and excess fresh denatured driver cDNA is added to perform the second hybridization. At the same time ,perform reverse-hybridization using astrocytomas grade I cDNA as tester and glioblastomas cDNA as driver. New hybrid molecules are formed which consist of differentially expressed cDNAs with different adaptors on each end.According to the princ...
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