RNA ElectrophoresisRNA Electrophoresis

Broad and Long Term ObjectiveBroad and Long Term Objective

To characterize the expression of ribulose 1-5 bisphosphate To characterize the expression of ribulose 1-5 bisphosphate carboxylase oxygenase and chlorophyll AB binding gene in carboxylase oxygenase and chlorophyll AB binding gene in Lycopersicon esculentumLycopersicon esculentum (Tomato) leaves subjected to 24, (Tomato) leaves subjected to 24, 48, and 72 hours in the dark48, and 72 hours in the dark

Research PlanResearch 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 ObjectivesToday’s Laboratory Objectives

1.1. To assess the integrity of the RNA extracted last To assess the integrity of the RNA extracted last week using agarose gel electrophoresis. week using agarose gel electrophoresis.

2.2. To prepare cDNA from total RNA using an To prepare cDNA from total RNA using an oligo dT primer oligo dT primer

3. To examine the success of the cDNA synthesis, and 3. To examine the success of the cDNA synthesis, and devise a strategy for examining the using realdevise a strategy for examining the using real time RT-time RT- PCRPCR

RNases ARE EVERYWHERE!RNases ARE EVERYWHERE!

Control of RNasesControl of RNases Wear Gloves and Practice Sterile TechniqueWear Gloves and Practice Sterile Technique Use Disposable Plastics or Baked GlasswareUse Disposable Plastics or Baked Glassware Use chemicals or reagents that will inactive RNAses (DEPC treated water, Use chemicals or reagents that will inactive RNAses (DEPC treated water,

chaotropic agents, etc)chaotropic agents, etc) Always Keep RNA on Ice or FrozenAlways Keep RNA on Ice or Frozen Work quickly and carefullyWork quickly and carefully

RNA ElectrophoresisRNA Electrophoresis

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

Electrophoresis of RNAElectrophoresis of RNA

Intact High Quality RNA Characterized by:Intact High Quality RNA Characterized by: Two prominent rRNA BandsTwo prominent rRNA Bands Slight smear of various sized mRNA molecules in backgroundSlight smear of various sized mRNA molecules in background

RNA Ladder 0.2-10 KbRNA Ladder 0.2-10 Kb

cDNA SynthesiscDNA Synthesis

cDNA is a DNA copy synthesized from mRNA.  The enzyme used is reverse transcriptase an RNA-dependent DNA polymerase isolated from a retrovirus (AMV or MMLV).  

Reverse TranscriptaseReverse Transcriptase

cDNA Synthesis ProductscDNA Synthesis Products

Northern Blot for Assessing Transcript Northern Blot for Assessing Transcript Size and Expression LevelSize and Expression Level

Ribonuclease Protection AssayRibonuclease Protection AssayRNA AbundanceRNA Abundance

Comparative/Quantitative RT- PCR Comparative/Quantitative RT- PCR (www.ambion.com)(www.ambion.com)

Real Time RT PCR Quantitative Technique for Measuring RNA Transcript Levels

Sample---RNA Isolation---cDNA Synthesis---RT PCR Amplification

Real Time PCR Work Flow

Defining Parameters ofDefining Parameters ofReal Time RT PCRReal Time RT PCR

Cycle Threshold: Cycle # when product fluoresence exceeds that of background

Fold Change: 2ΔCt

Melt Curve: fluorescence plotted as a function of temperature as thermal cycler heats through dissociate temperature of product

Comparative (Quantitative) real Comparative (Quantitative) real time Reverse Transcriptase PCRtime Reverse Transcriptase PCR

Next Week:

Assemble and run a real time RT PCR reaction using cDNA derived from RNA isolated from from Lycopersicon esculentumLycopersicon esculentum (Tomato) leaves from subjected to 24, 48, and 72 hours in the dark (Tomato) leaves from subjected to 24, 48, and 72 hours in the dark and RNA isolated from leaves under normal light conditions. and RNA isolated from leaves under normal light conditions. The expression of the RubisCO SS and CAB under these conditions will be examined.