Uskudar University
Molecular Cell Biology Laboratory
cDNA Synthesis & Real Time PCR
cDNA
• Complementary DNA (cDNA) is synthesised from an RNA template in a
reaction catalysed by the enzyme reverse transcriptase.
• cDNA synthesis is the first step in many molecular biology workflows,
such as gene expression studies using real-time PCR.
• cDNA is a DNA copy of a messenger RNA (mRNA) molecule produced by
reverse transcriptase, a DNA polymerase that can use either DNA or RNA
as a template.
• The main difference between DNA and cDNA is that DNA is composed
of both coding and non-coding sequences whereas cDNA only
contains the coding sequences.
• The coding sequences are the exons of a gene, which codes for a
functional protein. The non-coding sequences are the remaining DNA
sequences of the genome.
Real-Time PCR or Quantitative
PCR
• Real-time PCR is a sensitive technique used for
absolute and real-time quantification of nucleic
acids.
• It uses the same principle as conventional PCR
(denaturation, annealing, and extension) but also
uses a fluorescent DNA-binding dye (e.g., SYBR
Green) or probes (e.g., TaqMan probes) that emit
a fluorescent signal during amplification, thus
allowing detection and quantification of PCR
products in real time.
• Real-time PCR can enable quantification of small amounts of mRNA,
DNA, or cDNA in a biological sample.
• cDNA = complementary DNA (cDNA) is DNA synthesized from a single-
stranded RNA template in a reaction catalyzed by the enzyme reverse
transcriptase.
Types of Real-Time PCR
• There are currently three major methods of gene detection and quantification by real-time
PCR;
1. Real-time PCR using DNA-binding dyes (e.g., SYBR Green, SYTO 9, EVA Green, LC Green 1)
2. Real-time PCR using fluorescent primers (e.g., Amplifluor® system or LUX Fluorogenic
Primers)
3. Real-time PCR using fluorescent probes (e.g., TaqMan® probes)
Real-time PCR using DNA-binding dyes
(e.g., SYBR Green, EVA Green)
• The PCR is performed in the presence of a fluorescent dye which intercalates within a double-
strand DNA and emits fluorescence.
Real-time PCR using fluorescent primers
(LUX Fluorogenic Primers)
• For these experiments, a
fluorophore is attached directly to a
targetspecific primer. Thus, each
time the primer is used to synthesize
a new DNA strand (during annealing
and elongation), there is increase in
fluorescence.
Real-time PCR using fluorescent probes (e.g., TaqMan® probes)
• This procedure is very specific and very sensitive.
• It uses two target specific primers (forward and
reverse primers) and a probe.
Roche LightCycler® Nano Real-Time PCR
Probe 1μl
Primer (F) 0.5μl
Real time PCR Primer (R) 0.5μl
will be Buffer 2μl
performed in MgCl2 1.6μl
20 μl reaction dNTP 0.4μl
volume;
Ultra pure water 8.5μl
cDNA 5μl
Taq Polymerase 0.5μl
Initialization Denaturation
DNA
Amplification Annealing Extension
Steps
Final
Hold
Extension
• Ct Value?
• The Ct (cycle
threshold) is defined
as the number of
cycles required for
the fluorescent
signal to cross the
threshold.
• NTC?
• No template, no ct
expected.
The United States Centers for Disease Control and Prevention (CDC) uses a one-step real time
RT-PCR (rRTPCR) assay, which provides quantitative information on viral loads, to detect the
presence of SARS-CoV-2.
• To perform the assay, the viral RNA is extracted and added to a master mix.
The master mix contains nuclease-free water, forward and reverse
primers, a fluorophore-quencher probe, and a reaction mix (consisting of
reverse transcriptase, polymerase, magnesium, nucleotides, and additives).
• The master mix and extracted RNA are loaded into a PCR thermocycler, and
the incubation temperatures are set to run the assay.
• During rRTPCR, the fluorophore-quencher probe is cleaved, generating a
fluorescent signal. The fluorescent signal is detected by the thermocycler,
and the amplification progress is recorded in real time
