RNA Electrophoresis - CSUSM Home Page CSUSMRNA电泳-加利福尼亚州立大学圣马科斯分校主页加利福尼亚州立大学圣马科斯分校.pptVIP

RNA Electrophoresis Research Plan Today’s Laboratory Objectives 1. To assess the integrity of the RNA extracted last week using agarose gel electrophoresis. 2. To prepare cDNA from total RNA using an oligo dT primer 3. To examine the success of the cDNA synthesis, and devise a strategy for examining the using real time RT- PCR RNA Electrophoresis Electrophoresis of RNA Intact High Quality RNA Characterized by: Two prominent rRNA Bands Slight smear of various sized mRNA molecules in background RNA Ladder 0.2-10 Kb cDNA Synthesis Reverse Transcriptase cDNA Synthesis Products Northern Blot for Assessing Transcript Size and Expression Level Ribonuclease Protection AssayRNA Abundance Comparative/Quantitative RT- PCR () Comparative (Quantitative) real time Reverse Transcriptase PCR * * Broad and Long Term Objective To characterize the expression of ribulose 1-5 bisphosphate carboxylase oxygenase and chlorophyll AB binding gene in Lycopersicon esculentum (Tomato) leaves subjected to 24, 48, and 72 hours in the dark RNA Isolation leaf material grown in light and in the dark RNA Electrophoresis and cDNA synthesis Assessing Gene Expression Northern Blot RNase Protection Quantitative PCR Quantitative real time RT PCR RNases ARE EVERYWHERE! Control of RNases Wear Gloves and Practice Sterile Technique Use Disposable Plastics or Baked Glassware Use chemicals or reagents that will inactive RNAses (DEPC treated water, chaotropic agents, etc) Always Keep RNA on Ice or Frozen Work quickly and carefully RNA is highly susceptible to intrastrand H-bonding; such secondary structure will affects its migration through an agarose gel unless it is resolved Denaturing Agarose Gel Electrophoresis Two types: 1) Formaldehyde 2) glyoxyl dimethylsulfoxide Formamide and formalydehyde are included in Loading Buffer RNA samples heated at 65° C for 5 minutes prior to electrophoresis cDNA is a DNA copy synthesized from m