Download Real-time PCR

I. Real-time RT-PCR analysis of siRNA-induced
knockdown in mammalian cells
(Amit Berson, Mor Hanan and Dr. Mira Korner)
Applied Biosystems 7900HT Fast Real-Time PCR System
Experimental outline
1. We will use siRNA in order to knockdown the expression of two
prototypic splicing factors, SC35 and ASF/SF2
2. Examine the expression of the two splicing factors in HeLa cell line
after siRNA transfection using Real time RT-PCR
3. Furthermore, we will examine the expression of MKNK2 and
caspase9 splice variants. The alternative splicing of these genes
are known to be mediated by ASF/SF2.
Standard assays for RNA quantification
Gel based RT-PCR. RT to generate cDNA, PCR amplification for various number
of cycles and gel electrophoresis. The use of this method is rapidly decreasing with
the increasing popularity of real-time PCR. This method is both time-consuming and
to a great extent less accurate than real-time PCR
Northern blot – Direct visualization of the RNA with radioactive probe.
Micro-arrays – allows the comparison of many thousands of transcripts
between different samples. However, results must be validated with
conventional RT-PCR
Standard assays for RNA quantification
In-situ hybridization – the only method that detects RNA
molecules in their natural environment of the cell. This method is
less accurate for quantification but has the enormous advantage of
examining RNA levels in cell-type specific / intracellular localization
specific manner
Real-time PCR – allows monitoring of product accumulation in real-time
during the PCR process. This method has several major advantages:
accurate quantification, ability to distinguish between and primer-dimer or
a non-specific product, and easy handling and less time consuming
And more…. RNase protection etc.
Basics of real-time RT-PCR
Total RNA
Reverse transcription
using random or poly dT primers
cDNA
Polymerase chain reaction (PCR)
using specific primers
Amplified product
Real-time PCR assays
Real-time PCR assays
Advantages:
• High specificity
(no primer dimers)
• Basic chemistry suitable
for all amplicons - only
primer design is needed
Disadvantages:
• Lower specificity
• Requires probe design
and synthesis or the use
of commercially available
ready made and tested
probes
• More expensive
fluorescence
Amplification curve
cycle
Threshold cycle (CT)
In the exponential phase, DNA is amplified by
approximately 2-fold in each cycle
fluorescence
Melting curve
temperature
1 degree
intervals
95ºC
derivative
Cool down
temperature
Primer design
• All primers must have similar Tm (Melting temp.) we use
Tm of 59º ±1
• Amplicons must be similar in length – we use approx.
80bp amplicons
• Primers are designed to anneal to an exon-exon junction
or to flank an intron, to avoid amplification of genomic
DNA
Forward primer
Reverse primer
exon
intron
exon
exon
exon
Normalization: Reference genes
•
Use of an appropriate standard to normalize results is essential as pipetting
errors and differences in starting material amounts may lead to
misinterpretation of the results
What is an appropriate standard?
Several genes are commonly used as reference genes. However, for
each study a specific suitable reference gene, or a combination of
several genes should be used. For example:
•β-actin
•18S RNA
•GAPDH
•Etc.
In addition, well to well variability is normalized using a
passive reference fluorescent molecule - ROX
Controls
Non template control : tests contamination of stock solutions
-RT : tests carry over of genomic DNA
Absolute Vs. relative quantification
Absolute quantification is performed using amplification of known amounts of RNA
and plotting a standard curve of CT Vs. log of quantity
Relative quantification allows comparison of mRNA levels between different
samples without the need of absolute quantification
Thermal program:
50º 2.00 UNG…
95º 10.00 to start the polymerase activity
95º 0.15 double strands disassociation
60º 1.00 annealing, elongation, and fluorescence detection
Melting curve
X40
Serial dilutions
• In order to validate that
changes in RNA concentration
result in appropriate change in
CT (threshold cycle) we
perform real-time PCR on
serial dilution of a sample
From this data we can
also deduce the primer
efficiency:
Eff = 10^(1/slope)
NR plasmid
y = -2.9718x + 38.626
R2 = 0.9944
30
25
Ct
20
15
10
5
0
0
2
4
6
log10 copies
8
10
1:1 < 1:2 <
1:4 < …
Primer dimer or non-specific amplicons effect can
be minimized, BUT their amplification makes
quantification inaccurate
Reading temp: 60º
Reading temp: 80º
No primer dimer
Primer dimer