v' 1'1

1
(No Transcript)
2
Real-Time RT-PCR (QPCR)
v. 1.1
3
Methods in Common Use for Detecting/quantifying
Transcripts

• 1. Northern blotting and in situ hybridization
• RNAse protection assays
• cDNA microarrays
• 4. Reverse transcription-polymerase chain
  reaction with end-point or real-time quantitation.

4
Quantitation by RT-PCR
Conventional
Real-time
RNA extraction
RNA extraction
Reverse Transcription
Reverse Transcription
cDNA to PCR rx
cDNA to PCR rx
PCR reaction
Electrophoresis/ Dot Blots/imaging
Real-time PCR
Image analysis
5
Configuring Real-time RT-PCR Assays
1. One enzyme/one tube. Tth polymerase has
intrinsic RT activity. (Roche uses Mn2)
2. Two enzymes/one tube. (Stratagene 1-step
QRT-PCR Master Mix Qiagen QuantiTect Probe
RT-PCR Kit (707/200 rxs)
3. Two enzymes/two tubes. AMV-RT or MMLV-RT H-,
Taq polymerase. ( Stratagene Brilliant SYBR Green
QPCR Master Mix Qiagen QuantiTect SYBR Green
PCR Kit (356/200 rx) ABI SYBR Green PCR Core
Reagents
6
Planning Your QPCR Measurements

• RNA extraction. Methods of extraction and
  purification, DNase treatment. Evaluate by
  photometry electrophoresis.
• Reverse transcription. 1 µ g total RNA using
  random hexamers, oligo-dT or sequence-specific
  primer stop rx with 95º/5 min then EDTA for
  storing at 4º up to 1 mo. cDNA is at 10 ng/µl.
• PCR amplification considerations. Primer design.
  a) Span splice junctions (or use DNase) b)
  Choose a small amplicon with minimal secondary
  structure c) similar Tms, ca. 55-60 avoid 3
  complementarity, etc. Dilute cDNA to reduce
  inhibition and amplification of non-specific
  products. Enzyme/hot start choices.
• Detection system. Intercalating dye or
  hybridizing probe. Lower primer conc. for SYBR
  Green I assays. Acquire data at lowest temp.
  melting non-specific products.
• Reagent mixes. Commercial (ABI, Stratagene,
  Qiagen, Bio-Rad) or home-made (with Tris, KCl,
  MgCl2, Triton X-100, pH 8.5).

7
Specificity with dsDNA dyes depends on RNA preps
On-column DNase
No DNase
M. Surpin
8
Ways of Achieving a Hot-start

• Mg2 in wax beads.
• 2. Assemble reactions in a cold block transfer
  to preheated thermal cycler.
• 3. Polymerase antibodies. ABI AmpliTaq Gold
• 4. Heat-labile chemical modification of
  polymerase. Roche FastStart Taq DNA polymerase.
• 5. Polymerase binding oligos. J. Mol. Biol.
  264268 1996.
• 6. Primers with hairpin-like structure. Nuc.
  Acids. Res. 2894 2000.

9
Chemistry Choices
SYBR Green I
TaqMan
10
Fluorescence ResonanceEnergy Transfer (FRET)
11
Taqman Probes are Hydrolysis Probes
Polymerization
TaqMan Probe
Forward Primer
R
Q
5
5
3
5
3
5
Reverse Primer
Reverse Primer
12
Displacement, Hydrolysis and Polymerization
Completed
R
Q
5
5
3
3
5
5
13
Molecular Beacons are Hybridization Probes
Annealing Meas.
14
When to Choose SYBR Green I (an intercalating dye)
1. Assays that do not require specificity of
probe based assays. Detection of 1,000s of
molecules. 2. General screening of transcripts
prior to moving to probe based assays. 3. When
the PCR system is fully optimized - no primer
dimers or non-specific amplicons, e.g. from
genomic DNA.
15
When to Use Fluorescent Probes hydrolysis or
hybridization
1. Sensitive assays - 100s of molecules. 2.
Allellic Discrimination Assays 3. Multiplex
Reactions - relative quantitation using an
internal housekeeping gene in the same
tube 4. Detection of rare transcripts. 5. Low
level pathogen detection. 6. Absolute
Quantitation - Standard Curve. 7. Relative
Quantitation using ??CT method - - no standard
curve
16
Primer, Probe and Amplicon Design using Primer
Express (ABI)
Primer Design

• Amplicon length of 50-150 bp shorter is better
• Less stable 3 end (GC rule No more than two
• Gs and Cs in the last 5 bases). No 3
  complement.

Probe Design

• Tm 10C higher than primers (68-70C)
• No G on the 5 end
• Use strand with more Cs than Gs

17
Melting Curve Analysis Can Show Specificity of
Reactions

84.3

75.0
18
Anatomy of an Amplification Plot.
Sample
?Rn
Threshold
Rn
No Template Control
CT
0
10
20
30
40
cycle number
19
Primer Matrix-TaqMan Assays

• Matrix of primers 50, 300, 900nM (probe 200nM,
• fixed amount of target)
• Looking to balance the effective Tms and maximize
  the efficiency lowest CT with highest ?Rn with
  low primer concentration

20
Primer Matrix

• Looking for lowest Ct with highest ?Rn

21
Probe Titration

• To conserve the probe reagent
• Probe _at_ 25, 50, 75, 100, 125, 150, 175, 200, 225
  250nM
• Looking for lowest ?Rn with no change in Ct

22
Absolute Quantitation

• Determine starting copy number or concentration
• Prepare RNA or DNA standards, carefully quantify

Quantifying pathogens Basic research in
transcription Quality control Gene therapy DNA
damage
23
Relative Quantitation

• To determine fold differences of nucleic
• acids with statistical confidence.

• Usually RNA targets

• Gene expression
• Drug therapy

24
Absolute or Relative Standards

• Generate standard curves for Target gene
  (and Endogenous reference gene) using standards
  or RNA from a Calibrator tissue

Log ng Calibrator RNA (or copy number)
25
Relative Standard Curve

• 1. Generate CTs for dilution series of RNAs
  (cDNAs) for target and endog. Ref. RNAs from
  calibrator and experimental tissues.

2. Generate standard curves with CTs of target
and ref. RNAs from calibrator tissue. Calculate
target and ref. RNA amounts in experimental
tissue by interpolation.
3. Normalize target gene expression with ref.
gene expression for control and treated
experimental tissue, e.g. ng Target RNA/ng
Reference RNA.
4. Calculate ratios of Target RNAs in control
and experimental tissues.
Endog. Ref. ß-actin, GAPDH, rRNA, total RNA,
cell number
26
Relative Quantitation
Using The Standard Curve Method
Covered in ABI User Bulletin 2
27
Comparative CT Method ?? CT
28
Dynamic Range of the Assay
X1/X2 (1E)-?CT
? CT CT (X1)-CT(X2)
X2
X1
10ng
0.001ng
CT(X1)
CT(X2)

• Titrate a template 10, 1, 0.1, 0.01, 0.001 ng

29
Effect of Amplification Efficiency
Xn X0(1E)n
Case 1 E 0.9
Case 2 E 0.8
Xn 100 (10.9)30
Xn 100 (10.8)30
Xn 2.3 x 1010
Xn 4.6 x 109
Result A difference of 0.1 in
amplification efficiencies created a 5-fold
difference in the final ratio of PCR products
after 30 cycles.
30
Comparative CT Method ?? CT

• CT (Target gene, control) CT (Endog. refer.
  gene, control) ?CT,cont (Control tissue)
• CT (Target gene, exp.) CT (Endog. refer. gene,
  exp.) ?CT,exp (Experimental tissue)

DCT,cont DCT, exp ?? CT
31
Target RNA TNF? in Control tissue CTs -
25.645 - A12 25.971 - B12 Endogenous
reference 18s rRNA in Control tissue
CTs -13.666 - A12 13.513 - B12
Ave ?CT Control CT(target)-CT(end.ref.)
12.218
32
Target RNA TNF-? Experimental tissue CTs
21.476 - C12 21.274 -
D12 Endogenous reference RNA 18s rRNA
Experimental tissue CTs 13.481 - C12
13.478 - D12 Ave. ?CT(Exper.) CT (target) - CT
(end.ref.) 7.89
33
Relative Quantitation by ??CT Method
?CT (Control tissue) 12.218 ?CT
(Experimental tissue) 7.895 ??CT
?CT(Cont.) - ? CT(Exp.) 4.323 ??CT
log2(Targetexp/Targetcont) Relative fold
difference of expression of TNF-? in Experimental
tissue relative to Control tissue 2??CT
24.323 20-fold increase
34
Melting Curve Analysis Can Show Specificity of
Reactions

84.3

75.0
35
Melting Curve Analysis used in 7700 for SNP
Genotyping
Nature Biotech. 17 87, 1999
36
Home-made QPCR Mix
10X PCR Buffer 100 mM Tris/500 mM KCl, pH 8.5
(rm temp)
PCR Mix 2X Master Mix, F R primers, water QPCR
reactions 20 µl PCR mix 5 µl template
37
Amplification of plasmid with Home-made and
Bio-Rad QPCR SYBR Green mixes
Home-made
Bio-Rad
38
(No Transcript)
39
Data Analysis AlgorithmMulti-Componenting
Intensity
Intensity
Wavelength, nm
Wavelength, nm