RT PCR (APPLIED BIOSYSTEM
7000 SDS)
Afsheen Arif, PhD.
RT PCR (APPLIED BIOSYSTEM 7000 SDS)
SDS detection system software
WHAT TYPE OF INSTRUMENTS ARE USED WITH
REAL-TIME PCR?
Real-time PCR instruments consist of TWO main
components:
Thermal Cycler (PCR machine)
Optical Module (to detect fluorescence in the
tubes during the run)
WHAT IS REAL-TIME PCR USED FOR?
Real-Time PCR has become a cornerstone of molecular biology:




Gene expression analysis
 Cancer research
 Drug research
Disease diagnosis
 Viral quantification
Food testing
 Percent GMO food
Transgenic research
 Gene copy number
ISOLATION OF RNA

RNA isolation based on 3 principals
 Lysing of cells to release nucleic acids
 Inactivation of RNases
 Separation of RNA from DNA




Old School
 Tissue Homogenization (Grinding on Dry Ice/LN2)
 Hot Phenol/SDS or Guanidium thiocyanate
 CsCl gradient centrifugation
New
 Chaotropic Salt Isolation (Qiagen)
 Trizol (Invitrogen)
Isolation of mRNA from total RNA (oligo dT)
Isolation of cytoplasmic RNA
RNA QUALITY

Widely used approaches :





ratio A260/280; between: 1.9 to 2.1 ( 100 % purity)
Gel analysis of ribosomal RNA; intact 18S and 28S (doubling intensity on gel
of 28S compared to 18S).
Agilent bio-analyzer/ BioRad experion microfluidic
Nanodrop
Free of contamination :



DNA
Proteins
Inhibitors: carry over chemicals from purification steps
These contamination may depends on the tissue or cell
from which RNA come from.
REAL TIME PCR: GENERALITIES

The real-time polymerase chain reaction uses fluorescent reporter dyes
to combine DNA amplification and detection steps in a single tube
format.

Increase in fluorescent signal is recorded during the assay.

Fluorescent signal is proportional to the amount of DNA synthesized
during each amplification cycle.




Individual reactions are characterized by the cycle fraction at which
fluorescence first rises above a defined background fluorescence, a
parameter known as the threshold cycle (Ct) or crossing point (Cp).
Consequently, the lower the Ct, the more abundant the initial target.
The homogeneous format eliminates the need for post-amplification
manipulation and significantly reduces hands-on time and the risk of
contamination.
THREE MAIN CHEMISTRIES ARE USED IN REAL TIME PCR

Intercalating dyes
SYBR-Green: this dye fluoresce upon light excitation when bound to double
stranded DNA.


Advantage
Cost effective.
Easily added to assays
Amplification products can be verified by the use of melting curves.
one Ct or so more sensitive than probe-based assays.
Disadvantage:
Lack of specificity and fluorescence varies with amplicon length.
http://www.scitopics.com/Real_time_Polymerase_Chain_reaction.html
THREE MAIN CHEMISTRIES ARE USED IN REAL TIME PCR

Intercalating dyes
SYBR-Green
THREE MAIN CHEMISTRIES ARE USED IN REAL TIME PCR
Intercalating dyes
SYBR-Green
THREE MAIN CHEMISTRIES ARE USED IN REAL TIME PCR

Fluorophores attached to primers




Invitrogen's Lux or Promega's Plexor primers.
Advantage: Relatively inexpensive and amplification products
can be verified by melting curves.
Specificity depends on the primers.
Usually company-specific design software needs to be used
for optimal performance.
THREE MAIN CHEMISTRIES ARE USED IN REAL TIME PCR

Hybridisation-probe based methods: TaqMan or Molecular Beacons.
Most specific, as products are only detected if the probes hybridize to
the appropriate amplification products.
Many variations on this theme, with melt curve analysis possible for
some chemistries (UBI).
Main disadvantages are cost, complexity and occasional fragility of
probe synthesis.
There are potential problems associated with the fact that probebased assays do not report primer dimers that can interfere with the
efficiency of the amplification reaction.
TAQMAN PROBES: HOW IT WORKS





Oligonucleotides that contain a fluorescent dye, typically on the 5'
base, and a quenching dye, typically located on the 3' base.
When irradiated, the excited fluorescent dye transfers energy to the
nearby quenching dye molecule rather than fluorescing, resulting in a
nonfluorescent substrate.
TaqMan probes are designed to hybridize to an internal region of a PCR
product.
During PCR, when the polymerase replicates a template on which a
TaqMan probe is bound, the 5' exonuclease activity of the polymerase
cleaves the probe.
This separates the fluorescent and quenching dyes and FRET no longer
occurs. Fluorescence increases in each cycle, proportional to the rate
of probe cleavage.
TAQMAN PROBE
IMAGINING REAL-TIME PCR
MEASURING
QUANTITIES
How can all this be used to measure DNA
quantities??
What if YOU started with FOUR times as much
DNA template as I did?
I have 1,000,000 copies at cycle 25.
You have 4,000,000 copies!
So… You had 2,000,000 copies at cycle 24.
And… You had 1,000,000 copies at cycle 23.
IMAGINING REAL-TIME PCR
MEASURING
QUANTITIES
So… if YOU started with FOUR times as much
DNA template as I did…
Then you’d reach 1,000,000 copies exactly
TWO cycles earlier than I would!
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
IMAGINING REAL-TIME PCR
What if YOU started with EIGHT times LESS DNA template
than I did?
MEASURING
QUANTITIES
You’d only have 125,000 copies right now at cycle 25…
…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle
28 you’ll have caught up with 1,000,000 copies!
So… you’d reach 1,000,000 copies exactly THREE cycles
later than I would!
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
IMAGINING REAL-TIME PCR
MEASURING
QUANTITIES
We describe the position of the lines with a value that represents
the cycle number where the trace crosses an arbitrary
threshold.
This is called the “Ct Value”.
Ct values are directly related to the starting quantity of DNA, by
way of the formula:
Quantity = 2^Ct
5000000
4500000
4000000
Ct Values:
3500000
3000000
2500000
25
23
2000000
28
1500000
1000000
500000
0
0
5
10
15
20
25
30
35
40
HOW DO WE MEASURE DNA IN A PCR
REACTION?
3’
5’
5’
3’
Extension
ID
3’
5’
Taq
ID
ID
Taq
5’
ID
ID
3’
Apply Excitation
Wavelength
l
l
3’
l
ID
ID
ID
Taq
l
ID
ID
l
Repeat
Taq 5’
3’
WHAT TYPE OF SOFTWARE IS USED WITH
REAL-TIME PCR?
The real-time software converts the fluorescent signals in each well
to meaningful data.
1.
2.
3.
4.
1
Set up PCR protocol.
Set up plate layout.
Collect data.
Analyze data.
2
3,4
ACTUAL DATA


This is some actual data from a recent real-time PCR run.
Data like this can easily be generated by preparing a dilution series of DNA.
Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.
REAL-TIME DEMONSTRATION
Experimental Protocol


5000000
4500000

4000000
Standard curve with serial 10-fold dilutions
Two replicates on standard curve
Multiple unknown samples
3500000
3000000

2500000
2000000

1500000
25ul reactions
iQ SYBR Green Supermix (2x)
1000000
500000

0
0
5
10
15
20
25
30
35
40



940C 3 min
940C 30 sec
590C 60 sec
Go to Step 2- 40 Times
DEFINITION & TERMINOLOGY