Biology Lab Analysis

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Sub Aim 2.1. Generate global transcriptional sequencing data for CML and MLL cells to investigate the response of malignant cells to SMYD2 inhibitors. Experimental methods: Molecular analyses and RNA sequencing: We will constructed a total of four libraries for CML and MLL cell lines to reveal an overexpression of gene categories that involved in CML and MLL using RNA sequencing. We will compare between leukemia cells that have treated with one of SMYD2 inhibitor with untreated leukemia cell as a reference. 1. RNA Isolation: First we will extract the total RNA from CML and MLL cell lines using 10 mLTrizol reagent (Invitrogen) according to the manufacturer's recommendations. 2. RNA selection: PolyA+ RNA will be purified from 50µg total…show more content…
The amplification reaction will be performed at: 94°C for 30 sec, 64°C for 30 sec, followed by 68°C for 3 minutes. 7. Sequencing and data analysis: 10nM library will be used to prepare flowcells with approximately 30,000 clusters per lane. Cluster generation and sequencing will be performed using the Standard Cluster Generation kit and the 36 Cycle Solexa Sequencing kit on the Illumina Cluster Station and Illumina Genome Analyzer I following manufacturer's instructions. Sub Aim 2.2. Determine whether p53 is methylated in CML and MLL cell lines during SMYD2 inhibitors treatment. Immunoprecipitation (IP) and Western blotting: we will use this method for selectively the endogenous target protein p53 from a complex proteins solution. For prepare the lysate, CML and CLL cells with and without SMYD2 inhibitors will be harvested and then lysed in 1 mL Lysis Buffer (5O mM Tris, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT) with protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). The lysates will be centrifuged and the supernatant will be incubated with p53 antibody bound to magnetic protein A beads for 2h. Then, p53 will be eluted from the beads using 100 µl of Elution Buffer. After that, immunoprecipitated protein will be resolved on SDS-PAGE and separated protein (p53) will be transferred to nitrocellulose (Protran BA, Schleicher and Schuell, NH), blocked using 5% nonfat milk (10 g nonfat
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