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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.
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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 realtime 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 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
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).
Northern Blot for Assessing Transcript Size and Expression Level
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 ofReal 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.
