Transcript RNA Electrophoresis

RNA Electrophoresis
Broad and Long Term Objective
To characterize the expression of metacaspase in E.
huxleyi cells grown in phosphate limiting media
versus nutrient rich media
Research Plan
RNA Isolation from cells grown in F/2 and F/50 media
RNA Electrophoresis
Assessing Gene Expression
Northern Blot
RNase Protection
Quantitative PCR
Today’s Laboratory Objectives
1. To assess the integrity of the RNA e
last week using agarose gel
electrophoresis.
extracted
2. To make cDNA from total RNA using Reverse
Transcriptase and an oligo dT primer
3. To assess the success of the cDNA synthesis reaction
and devise a strategy using real time RT PCR to examine
the expression of metacaspase in cells grown in
phosphate limiting and phosphate replete media
CAUTION: 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
Expected Results


Two prominent rRNA Bands
Slight smear of various sized
mRNA molecules in
background
Ribonuclease Protection Assay (www.ambion.com)
Comparative (Quantitative) RTPCR (www.ambion.com)
Northern Blot
Real Time RT PCR to Validate Differentially
Expressed Genes Reveal by SSH
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