Analysis of Gene Expression by Real Time RT 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, 28, and 72 hours in the dark To characterize the expression of ribulose 1-5 bisphosphate carboxylase oxygenase and chlorophyll AB binding gene in Lycopersicon esculentum (Tomato) leaves subjected to 24, 28, and 72 hours in the dark

Research Plan 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

Today’s Laboratory Objectives 1. To isolate high quality total RNA from tomato leaf tissue (growth conditions include light and 24, 48, or 72 hr dark) 1. To quantitate the amount and purity of the total RNA isolated 2. To become familiar with the nuances in handling RNA

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 Extraction Procedure Lyse Cells and Inhibit RNAses Harvest Tissues Separate RNA from Other Cellular Macromolecules Precipitate RNA

Guanidinium Isothiocyanate Extraction Cell Lysis  Cells harvested (1 gm)  Cell lysis accomplished via grinding in liquid nitrogen  Guanidinium thiocyanate= chaotropic agent inhibits RNases  Sarkosyl= disrupts membranes and inhibits ribonucelases  B-mercaptoethanol= reduces disulfide bonds of RNAses and prevents free-radical crosslinking of phenolics to DNA

Phenol Chloroform Extraction Phenol pH 4.3 Hydroylzes DNA Denatures Proteins Complexes Lipids/CHOs While RNA remains in Aqueous Phase

Precipitation of RNA Principle: Need enough salt to neutralize the repulsion among the negatively charged strands of DNA, and enough alcohol to pull water molecules out of the DNA strand. Lithium Chloride- selectively precipitates RNA Isopropanol more effective for smaller amounts of nucelic acids

UV Spectrophotometry for Quantitating Amount and Purity of RNA To Quantify your RNA sample: A260 x Dilution Factor x 40 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlength A260 x Dilution Factor x 40 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlength To estimate the purity of your sample: A260/A280= ratio of nucleic acids/protein A260/A280= is optimal for RNA

Next Week RNA Electrophoresis RNA ElectrophoresiscDNA Synthesis