RNA Electrophoresis
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
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 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 RTPCR
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 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 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
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 Transcriptase
cDNA Synthesis Products
Northern Blot for Assessing Transcript
Size and Expression Level
Ribonuclease Protection Assay
RNA Abundance
Comparative/Quantitative RT- PCR
(www.ambion.com)
Real Time RT PCR Quantitative Technique for
Measuring RNA Transcript Levels
Real Time PCR Work Flow
Sample---RNA Isolation---cDNA Synthesis---RT PCR Amplification
Defining Parameters of
Real 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
time Reverse Transcriptase PCR
Next Week:
Assemble and run a real time RT PCR reaction using cDNA
derived from RNA isolated from Lycopersicon esculentum
(Tomato) leaves from subjected to 24, 48, and 72 hours in the dark
and RNA isolated from leaves under normal light conditions. The
expression of the RubisCO SS and CAB under these conditions
will be examined.