Download Analysis of Real Time RT PCR Data

Analysis of gene expression by
real time PCR
Analysis of real time PCR data
Broad and Long Term Objective
To characterize the expression of the genes encoding
Ribulose 1-5 bisphosphate carboxylase oxygenase-3
(RBCS3) and Chlorophyll A/B binding protein-1b (Cab1b) in the leaves of tomato plants grown under
continuous light or subjected to 24 hr, 48 hr, or 72 hr in
complete darkness.
Research Plan
RNA Isolation from tomato leaves (continuous light or
24-72 hr darkness)
RNA Electrophoresis
cDNA synthesis
RBCS3 and Cab-1b transcript quantitation
by real time PCR
Analysis of real time PCR data
Today’s Laboratory Objective
To analyze your data output from the iCycler,
allowing you to determine:
• Specificity of RBCS3 or Cab-1b amplification
• Relative quantitation of RBCS3 or Cab-1b
transcript levels in light- vs. dark-grown tomato
plants
Output data: Amplification plot*
C1
B1
Ct Table*
Ct
Identifier
25.9
B1 (red)
27.0
C1 (blue)
Etc.
Threshold
Relative fluorescence units
Output data: Amplification plot
Wednesday Lab, Group #9
Operator variability in real time
PCR
Relative quantitation of transcript
levels*
• Determine average Ct for each treatment (light, dark)
± standard deviation
• Determine ΔCt |Ct dark – Ct light|
• Calculate relative difference in transcript levels between
samples (2ΔCt)
• Make a histogram to describe the relative difference in
transcript abundance between light
and dark samples
Caveats:
• Amplification efficiency is rarely 2
• Genomic DNA contamination
• No internal standard transcript
Output data: Melt curve*
Gradual temperaturedependent fluorescence
quenching
Rapid decrease in
fluorescence caused by
denaturation of dsDNA
(PCR product)
Melt peak* (85.5º C)
Relative fluorescence units
Negative first derivative of
fluorescence/temperature
Identification of multiple PCR
products using a melt curve
product 1
primer dimer
(Tm 85.5º C)
(Tm 77.0º C)
Under identical solvent
conditions, Tm is determined
by G/C content and length of
dsDNA
product 2
(Tm 86.9º C)
Chlorophyll A/B Binding Protein (CAB-1b)
Accession # M14443 Coding Sequence
1
61
121
181
241
301
361
421
481
541
601
661
721
781
atggcagctg
tcaccatctg
aagtccgccc
ttctctggtg
accgctggac
cactgcagat
aatggtgtca
ggtggacttg
tgggcttgcc
cttggtgagg
gaagacccag
atgttctcta
aaccttgctg
tttgtccccg
Start
ctacaatggc
cctcagaaat
catctagcag
agtccccaag
tttcagcaga
gggctatgct
agttcggtga
actacttggg
aagttgtgtt
ttgtcgaccc
aggcatttgc
tgtttggatt
atcaccttgc
gaaaatga
tctttcttcc
ttctggaaat
cccatggtat
ctacttgacc
ccctgaaact
tggtgctctt
ggctgtgtgg
caacccaagc
gatgggagct
actctaccct
tgagctcaag
ctttgttcaa
agaccccgta
Length
ccttcatttg
ggaaggatca
ggccctgacc
ggtgaatttc
tttgccaaga
ggatgtgtct
ttcaaggccg
ttggtccatg
gttgagggtt
ggtggcagct
gtaaaggaga
gctattgtca
aacaacaatg
ctggacaggc
ctatgagaaa
gtgttaagta
ctggtgatta
accgtgaact
tccctgagct
gatcccagat
cacaaagcat
accgtattgc
tcgacccatt
tcaagaacgg
ccggaaaggg
cctgggcttt
agtcaaactc
ggctgttgcc
cttgggccca
cgggtgggat
tgaagtgatc
cttggcccgt
cttcagtgaa
cttggccatc
tggtggacct
aggccttgct
tagacttgct
tccattggag
cgccacaaac
Tm
%G+C
Seq
59.98
59.68
58
50
ggccctgaccgtgttaagt
ttggtgctcttggatgtgtc
Cab primers
Left Primer
Right Primer
Product size
151
339
19
20
189
52
Rubisco Small Subunit (RBCS3)
Accession #X05986 Coding Sequence
1
61
121
181
241
301
361
421
481
atggcttctt
agcatggttg
caaaaccttg
tggccaccaa
gagcaattgc
ttcgagactg
ggcagatact
ttggctgagg
ttcgacaatg
cagtaatgtc
cacccttcac
acattacctc
ttaacatgaa
tcagcgaaat
agcacggatt
ggaccatgtg
tgcaggaggc
ttcgtcaagt
ctcagcagct
tggactcaag
cattgctagc
gaagtacgag
tgagtaccta
tgtgtaccgt
gaagttgccc
aaagaaggct
gcagtgcatc
gttgccaccc
tccaccgctt
aacggtggaa
actctgtcgt
ttgaaaaatg
gagaaccata
atgttcgggt
tacccacagg
agtttcatcg
gcggcaatgg
ctttccctgt
gagtcagttg
accttcctga
gatgggttcc
agtcaccagg
gcactgatgc
catgggtccg
cttacaagcc
tgcacaagct
ttcaaggaag
catgcaggtg
tttgtccgac
ttgcttggaa
atactacgat
aacccaggtc
tatcatcgga
cgaaggatac
541 taa
Start
Length
Tm
%G+C
Seq
Rubisco Set A
Left Primer
Right Primer
Product Size:
Rubisco Set B
Left Primer
Right Primer
Product Size:
276
422
20
20
147
60.69
59.58
45
50
50
AAATGGATGGGTTCCTTGCT
AAGACCTGGGTTGCATCAGT
216
375
22
20
160
59.87
59.96
50
55
47
GTCGTACCTTCCTGATTTGTCC
GGTCCAGTATCTGCCATCGT
Expected product melting
temperatures*
Melting temperature formula, dsDNA
Tm (ºC)= 81.5 + 16.6 log10 ([Na+]/{1.0 + 0.7[Na+]}) + 0.41(%[G+C]) – 500/n – 1(% mismatch)
% GC
Product
0
length (no mismatch)
K+, not Na+ is primary cation in the PCR reaction
[K+] = 0.0284 M in the PCR reaction
for this melting equation, this is equivalent to 0.114 M Na+
Calculated and observed melting temperatures may vary by ± 5º C, due
to variations in salt concentration and specific base composition
Next Week
• Lab report 3 due Wednesday, Dec. 14,
5:00 pm
• Rubisco (RbcS), chlorophyll a/b-binding
protein (CAB or Lhc), tomato
• Good luck with finals
• Have a nice winter break