Real Time RT PCR

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Real Time RT PCR
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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 either 48
or 72 hours in the dark as compared to the
expression in leaves grown under normal 12 hr
light/dark cycle and harvested at noon.

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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
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Electrophoresis of RNA and cDNA

• Intact High Quality RNA Characterized by
• Two prominent rRNA Bands
• Slight smear of various sized mRNA molecules in
  background
• When isolating RNA from leaf material, may see
  plastid rRNA bands
• cDNA Synthesis Products Characterized by
• A smear of many different sizee transcripts
  between .5-3.0 Kb

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Todays Laboratory Objectives

• To understand the theoretical basis behind
  primer design for real time RT-PCR analysis
• To become familiar with the manner in which a
  real time RT PCR experiment is set up and the
  data is collected

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Primer Design

• Primer Design Parameters

• Targets an amplicon length of 75 to 150 bp
• 50 to 60 GC content
• Limit secondary structure
• Limit stretch of G or Cs longer than 3 bases
• No stable interaction between forward and reverse
  primers
• Place Cs and Gs on ends of primers (no more
  than 2 inlast 5 bases on 3 end)
• Melting Temperature (Tm) above 50 C (70-72 C)
• Verify specificity

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Experimental Design

• Relative Expression
• Samples 1) cDNA derived from RNA from leaves
  at 48 or 72 hour in dark
• 2) cDNA derived from RNA from leaves
  harvested at 12 noon
• 3) no template control
• Each experimental sample assayed in triplicate
• No template controls assayed in duplicate
• Total of 8 samples/group
• Dilute cDNA 1/100
• Prepare a Master Mix for 9 Rxns

Reaction Mix cDNA (diluted 1100) 5.0 µl Sybr
Green Super Mix 7.1 µl x 9 63.9 ul Primer A
(20 µM) 0.3 µl x 9 2.7 ul Primer B (20
µM) 0.3 µl x 9 2.7 ul dH2O
12.3 µl x 9 110.7 ul total volume per reaction
25 µl x 9
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Plate Set Up
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RubisCO Small Subunit accession X05986 Coding
Sequence
1 aaaaatgaaa aactcgtcag aaagaaaaag caaaagcaac
aaaaaaattg caagtatttt 61 ttaaaaaaga aaaaaaaaac
atatcttgtt tgtcagtatg ggaagtttga gataaggacg 121
agtgaggggt taaaattcag tggccattga ttttgtaatg
ccaagaacca caaaatccaa 181 tggttaccat tcctgtaaga
tgaggtttgc taactctttt tgtccgttag ataggaagcc 241
ttatcactat atatacaagg cgtcctaata acctcttagt
aaccaattat ttcagcaatg 301 gcttcttcag taatgtcctc
agcagctgtt gccacccgcg gcaatggtgc acaagctagc 361
atggttgcac ccttcactgg actcaagtcc accgcttctt
tccctgtttc aaggaagcaa 421 aaccttgaca ttacctccat
tgctagcaac ggtggaagag tcagttgcat gcaggtttgt 481
gtgtgtatat atatatacgt acaacaaaat tcattgacta
taatgttata ctcgattagc 541 taatttaact atttataatt
gtataggtgt ggccaccaat taacatgaag aagtacgaga 601
ctctgtcgta ccttcctgat ttgtccgacg agcaattgct
cagcgaaatt gagtacctat 661 tgaaaaatgg atgggttcct
tgcttggaat tcgagactga ggtcaacatc tatctcctct 721
gtttttaaaa tttactagct agtatgttga tatgtcgtgt
taacagtgtt gtgggatatc 781 atgtgcagca cggatttgtg
taccgtgaga accataagtc accaggatac tacgatggca 841
gatactggac catgtggaag ttgcccatgt tcgggtgcac
tgatgcaacc caggtcttgg 901 ctgaggtgca ggaggcaaag
aaggcttacc cacaggcatg ggtccgtatc atcggattcg 961
acaatgttcg tcaagtgcag tgcatcagtt tcatcgctta
caagcccgaa ggatactaaa 1021 tgtgtatatg tcaacagtga
gaaactgttc gcattttccg ttttgcttct ttctttctat 1081
tcaatgtatg ttgttggatt ccagttgaat ttattatgag
aactaataat aatagtaata 1141 atcatttgtt tctttactaa
tttgcatttt cacatatgat ttctggtgca tatcataatt 1201
ttcattccac caatattaat ttccccattc aagttactta
tgaaatagaa atcctcttct 1261 ccgactactt tatttgtccg
aaagtcttgt ggctgctata taacgcaaaa tggatagaga 1321
agattcatta ctaagccgat c
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RubisCO Primer Sets

• Start Length Tm GC Seq
• Primer Set A
• Left Primer 276 20 60.69 45
  AAATGGATGGGTTCCTTGCT
• Right Primer 422 20 59.58
  50 AAGACCTGGGTTGCATCAGT
• Product Size 147
• Primer Set B
• Left Primer 216 22 59.87 50 GTCGTACCTTCCTGATT
  TGTCC
• Right Primer 375 20 59.96 55 GGTCCAGTATCTGCCA
  TCGT
• Product Size 160

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Chlorophyll A/B Binding Protein (CAB-1b)
Accession M14443 Coding Sequence

• 1 atgaagaagt tgatggatta tagattgcca
  agtgtgctac acatgggatc ttgataccca
• 61 atgagatcat acatatagat atcacttgat
  aagatgattc tctctctttt ctcctatata 121 ttctcaaccc
  caactaactt catcttcatc acccatcaaa cacttaattc
  ttctcttaaa 181 ataaacacaa atggcagctg ctacaatggc
  tctttcttcc ccttcatttg ctggacaggc 241 agtcaaactc
  tcaccatctg cctcagaaat ttctggaaat ggaaggatca
  ctatgagaaa 301 ggctgttgcc aagtccgccc catctagcag
  cccatggtat ggccctgacc gtgttaagta 361 cttgggccca
  ttctctggtg agtccccaag ctacttgacc ggtgaatttc
  ctggtgatta 421 cgggtgggat accgctggac tttcagcaga
  ccctgaaact tttgccaaga accgtgaact 481 tgaagtgatc
  cactgcagat gggctatgct tggtgctctt ggatgtgtct
  tccctgagct 541 cttggcccgt aatggtgtca agttcggtga
  ggctgtgtgg ttcaaggccg gatcccagat 601 cttcagtgaa
  ggtggacttg actacttggg caacccaagc ttggtccatg
  cacaaagcat 661 cttggccatc tgggcttgcc aagttgtgtt
  gatgggagct gttgagggtt accgtattgc 721 tggtggacct
  cttggtgagg ttgtcgaccc actctaccct ggtggcagct
  tcgacccatt 781 aggccttgct gaagacccag aggcatttgc
  tgagctcaag gtaaaggaga tcaagaacgg 841 tagacttgct
  atgttctcta tgtttggatt ctttgttcaa gctattgtca
  ccggaaaggg 901 tccattggag aaccttgctg atcaccttgc
  agaccccgta aacaacaatg cctgggcttt 961 cgccacaaac
  tttgtccccg gaaaatgact ctaaacgtct caagtcttgg
  tcgtttgatg
• 1021 acagtgtaaa gatgtagtgt gctacctgac
  aatataatga aattttgttt gtgtttgaat
• 1081 ggcttttctg tactgagttt cattttccca
  agtcaactca taaatcaagc actaacaatg
• 1141 atacaacaaa atgacccctc acatatgagt
  aataactaga aaaactgcaa tgctatgttg
• 1201 taaggttgaa cttgaatttt caactagagc
  agtttattta atttaatgaa ttc

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Chlorophyll A/B Binding Protein Primer Set

• Start Length Tm GC Seq
• Primer Set A
• Left Primer 55 21 59.86 47.62
  AAACTCTCAACCATCTGCCTCA
• Right Primer 236 20 60.74
  50 CACCCGTAATCACCAGGA
• Product Size 147

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Real Time RT PCR
Real Time PCR Work Flow
Sample---RNA Isolation---cDNA Synthesis---RT PCR
Amplification
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Real Time RT PCR Cycling Parameters

• Polymerase activation 95 C 10 min
• 40 cycles
• Denaturation 95 C 60 sec
• Primer Annealing 60 C 30 sec
• Extension 72 C 45 sec
• Melt Curves
• Denaturation 95 C 1 min
• Renaturation 55 C 1 min
• Denaturation Ramp 0.5 C every 10 sec

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Defining Parameters ofReal Time RT PCR
Cycle Threshold Cycle when product
fluoresence exceeds that of background
Fold Change 2?Ct
Melt Curve fluorescence plotted as a
function of temperature as thermal cycler heats
through dissociate temperature of product
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Presentation of Real Time RT PCR Data

• What to include
• Melt Curve
• Raw Quantitative Graph
• Histogram of Relative Fold Change

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Next Lab Dec 5th

• Real Time RT PCR Data Analysis using ICycler
  Software