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Basic Principles of RT-qPCR
Introduction to RT-qPCR
RNA as the Starting Material
Quantitative reverse transcription PCR (RT-qPCR) is used when the starting material is RNA. In this method, RNA is first transcribed into complementary
DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the qPCR reaction. RTqPCR is used in a variety of applications including gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing,
and disease research.
One-step vs. Two-step RT-qPCR
RT-qPCR can be performed in a one-step or a two-step assay (Figure 1, Table 1). One-step assays combine reverse transcription and PCR in a single
tube and buffer, using a reverse transcriptase along with a DNA polymerase. One-step RT-qPCR only utilizes sequence-specific primers. In two-step
assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming
strategies.
Click image to enlarge
Figure 1. One-Step vs. Two-Step RT-qPCR.
Table 1. Advantages and Disadvantages when using one-step versus two-step assays in RT-qPCR
Advantages
One-step
Two-step
Disadvantages
Less experimental variation since both reactions take
Impossible to optimize the two reactions separately
place in the same tube
Less sensitive than two-step because the reaction
Fewer pipetting steps reduces risk of contamination
conditions are a compromise between the two combined
Suitable for high throughput amplification/screening
reactions
Fast and highly reproducible
Detection of fewer targets per sample
A stable cDNA pool is generated that can be stored for
The use of several tubes and pipetting steps exposes
long periods of time and used for multiple reactions
the reaction to a greater risk of DNA contamination
The target and reference genes can be amplified from the
Time consuming
same cDNA pool without multiplexing
Requires more optimization than one-step
Optimized reaction buffers and reaction conditions can be
used for each individual reaction
Flexible priming options
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Reverse Transcription in RT-qPCR
Choosing total RNA vs. mRNA
When designing a RT-qPCR assay it is important to decide whether to use
total RNA or purified mRNA as the template for reverse transcription.
mRNA may provide slightly more sensitivity, but total RNA is often used
because it has important advantages over mRNA as a starting material.
First, fewer purification steps are required, which ensures a more
quantitative recovery of the template and a better ability to normalize the
results to the starting number of cells. Second, by avoiding any mRNA
enrichment steps, one avoids the possibility of skewed results due to
different recovery yields for different mRNAs. Taken together, total RNA is
more suitable to use in most cases since relative quantification of the
targets is more important for most applications than the absolute sensitivity
of detection1.
Primers for Reverse Transcription
Three different approaches can be used for priming cDNA reactions in
two-step assays: oligo(dT) primers, random primers, or sequence specific
primers (Figure 2, Table 2). Often, a mixture of oligo(dT)s and random
primers is used. These primers anneal to the template mRNA strand and
provide reverse transcriptase enzymes a starting point for synthesis.
Figure 2. Four different priming methods for the reverse transcription step in two-step
assays of RT-qPCR.
Table 2. Primer considerations for the cDNA synthesis step of RT-qPCR. Combining random primers and
anchored oligo(dT) primers improves the reverse transcription efficiency and qPCR sensitivity.
Primer Options
Structure and Function
Advantages
Oligo(dT)s (or
Stretch of thymine residues that anneal to
Generation of full length cDNA
Only amplify gene with a
anchored
poly(A) tail of mRNA; anchored
from poly(A)-tailed mRNA
poly(A) tail
oligo(dT)s)
oligo(dT)s contain one G, C, or A (the
Good to use if little starting
Truncated cDNA from
anchor) residue at the 3′ end
material is available
priming internal poly(A)
Anchor ensures that the
sites*2
oligo(dT) primer binds at the 5′
Bias towards 3′ end*
end of the poly(A) tail of mRNA
Disadvantages
*Minimized if anchored oligo(dT)s
are used
Random
Six to nine bases long, they anneal at
Anneal to all RNA (tRNA, rRNA,
cDNA is made from all RNAs
Primers
multiple points along RNA transcript
and mRNA)
which is not always desirable
Good to use for transcripts with
and can dilute mRNA signal
significant secondary structures,
Truncated cDNA
or if little starting material is
available
High cDNA yield
Sequence
Custom made primers that target specific
Specific cDNA pool
Synthesis is limited to one
Specific Primers
mRNA sequence
Increased sensitivity
gene of interest
Use reverse qPCR primer
Reverse Transcriptase Enzymes
Reverse Transcriptase is the enzyme that makes DNA from RNA. Some enzymes have RNase activity to degrade the RNA strand in the RNA-DNA
hybrid after transcription. If an enzyme does not possess RNase activity, an RNaseH may be added for better qPCR efficiency. Commonly used enzymes
include Moloney murine leukemia virus reverse transcriptase and Avian myeloblastosis virus reverse transcriptase. For RT-qPCR, it is ideal to choose a
reverse transcriptase with high thermal stability, because this allows cDNA synthesis to be performed at higher temperatures, ensuring successful
transcription of RNA with high levels of secondary structure, while maintaining their full activity throughout the reaction producing higher cDNA yields.
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RNase H Activity of Reverse Transcriptase
RNase H activity degrades RNA from RNA-DNA duplexes to allow efficient
synthesis of double-stranded DNA. However, with long mRNA templates,
RNA may be degraded prematurely resulting in truncated cDNA. Hence, it
is generally beneficial to minimize RNase H activity when aiming to
produce long transcripts for cDNA cloning. In contrast, reverse
transcriptases with intrinsic RNase H activity are often favored in qPCR
applications because they enhance the melting of RNA-DNA duplex during
the first cycles of PCR (Figure 3).
Figure 3. RNase H Activity of reverse transcriptases. In qPCR, use a reverse
transcriptase with RNAse activity.
cDNA Synthesis in a Thermal Cycler
Step 1
Step 2
Step 3
Step 4
Predenaturation (Optional)
Primer Extension
cDNA Synthesis
Reaction Termination
This step is recommended if the RNA template has a high degree of secondary structure.
qPCR in RT-qPCR
Primer Design
PCR primers for the qPCR step of RT-qPCR should ideally be designed to
span an exon-exon junction, with one of the amplification primers
potentially spanning the actual exon-intron boundary (Figure 4). This
design reduces the risk of false positives from amplification of any
contaminating genomic DNA, since the intron-containing genomic DNA
sequence would not be amplified.
If primers cannot be designed to separate exons or exon-exon boundaries,
it is necessary to treat the RNA sample with RNase-free DNase I or
dsDNase in order to remove contaminating genomic DNA.
Controls for RT-qPCR
Click image to enlarge
Figure 4. Primer Design for the qPCR step of RT-qPCR. 1) If one primer is designed
A minus Reverse Transcription control (-RT control) should be included in
all RT-qPCR experiments to test for contaminating DNA (such as genomic
DNA or PCR product from a previous run). Such a control contains all the
reaction components except for the reverse transcriptase. Reverse
transcription should not occur in this control, so if PCR amplification is
to span an exon-intron boundary, the possible contaminating genomic DNA is not
amplified, because the primer cannot anneal to the template. In contrast, cDNA does
not contain any introns, and is efficiently primed and amplified. 2) When primers flank a
long (e.g. 1 kb) intron, the amplification cannot occur because the short extension time
is sufficient for the short cDNA sequence but not for the longer genomic target.
seen, it is most likely derived from contaminating DNA.
References
1. Bustin S. (ed) (2004) A-Z of Quantitative PCR. IUL Biotechnology Series, International University Line, La Jolla, California.
For Research Use Only. Not for use in diagnostic procedures.
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