Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ™ Real-Time PCR Detection System and iQ™ Supermix, Rev A
Jessica N Ebright1 and Catherine Bowes Rickman1, 2, 1Departments of
Ophthalmology and 2Cell Biology, Duke University Medical Center, Durham,
NC 27710 USA
Correspondence: Catherine Bowes Rickman, PhD, Department of Ophthalmology,
Duke University Medical Center, Box 3802, Durham, NC 27710, Phone (919)
668-0648, Fax (919) 684-3687, E-mail: bowes007@duke.edu
Introduction
We are interested in identifying genes that are differentially expressed
within the central (macular) region of the human retina. Expression profiles
of thousands of genes from this small, highly specialized region of the
central nervous system were obtained by comparative screening of human
cDNA microarrays with human macula- and mid-peripheral retina (periphery)-derived
RNAs. In order to validate the maculaenriched expression of genes identified
by our array analysis, we must rely on a PCR-based method of quantitation.
Realtime RT-PCR quantitates the initial amount of a template with more
specificity, sensitivity, and reproducibility than any other method. There
are many factors that contribute to the consistent performance of a real-time
quantitative RT-PCR assay, and many aspects that must be optimized when
putting this powerful technology to work in a new experimental system.
Methods
Total RNA was isolated from 4 mm trephine punches of neural retina from
two areas, the macula and the midperiphery, using Trizol reagent (Invitrogen)
with glycogen added as a carrier as described by Bracete et al. (1999)
with the following modification: 0.9 ml Trizol reagent plus 13.5 µl glycogen
(20 mg/ml, Roche Molecular Biochemicals) was added to flash-frozen tissue
in a 1 ml microcentrifuge tube and vigorously homogenized for 30 sec using
an Ultraturrax T8 homogenizer (Ika Laboratories). Total RNA was DNasetreated
using DNA-free reagent (Ambion), and RNA yields were determined by fluorescence
at 530 nm using RiboGreen RNA quantitation reagent (Molecular Probes)
as described by the manufacturer. First-strand cDNAs were synthesized
from equal amounts of total RNA (1 µg/reaction) using oligo(dT) primers
and SuperScript II reverse transcriptase (Invitrogen) according to the
manufacturer’s instructions.
Gene-specific primers (GSPs) were designed to anneal near the 3' end
of two mRNA transcripts and to generate PCR products 75–300 base pairs
long. Three GSP pairs amplify different overlapping regions of a single
transcript that is enriched in the macula, while one GSP pair detects
a human housekeeping gene transcript, β-actin (ACTB), which is constitutively
expressed in the neural retina. The amplified regions spanned exon-exon
junctions when possible. All primers were purchased from Proligo. RT-PCR
was performed using the GSP pairs in reactions amplifying across a gradient
of annealing temperatures to identify optimal reaction conditions for
real-time RT-PCR, and PCR product lengths were verified on a 4.5% Super
AcrylAgarose gel (DNA Technologies). Real-time quantitative RT-PCR was
performed using an iCycler iQ system (Bio-Rad). The rate of accumulation
of amplified DNA was measured by continuous monitoring of SYBR Green I
(Molecular Probes) fluorescence. Melt curves of the reaction products
were generated, and fluorescence data were collected at a temperature
above the melting temperature of nonspecific products (Morrison et al.
1998).
Specifically, quantitative real-time RT-PCR on the iCycler iQ was performed
in duplicate or triplicate on 1 µl of template cDNA per 20 µl reaction.
Mix A reactions consisted of PCR buffer (16.6 mM (NH4)2SO4, 67 mM Tris,
pH 8.8, 6.7 mM MgCl2, 10 mM β-mercaptoethanol; Loging et al. 2000), 1
mM dNTPs (Invitrogen), 0.5 U of Platinum Taq DNA polymerase (Invitrogen),
10 nM fluorescein calibration dye (Bio-Rad), 1 µl of a 1:1,500 dilution
of 10,000x SYBR Green I stock, 500 nM of each GSP, and 1 µl of cDNA. iQ
supermix reactions consisted of iQ supermix (Bio-Rad) at a final concentration
of 1x, 10 nM fluorescein calibration dye, 1 µl of a 1:1,500 dilution of
10,000x SYBR Green I stock, 500 nM of each GSP, and 1 µl of cDNA. To control
for pipetting losses, 19 µl of each 20 µl reaction was amplified in a
96- well thin-wall PCR plate (Bio-Rad) using the following PCR parameters:
95°C for 2 min followed by 50 cycles of 95°C for 15 sec, 60°C for 15 sec,
and 72°C for 15 sec. Melt-curve analysis was performed immediately following
amplification by increasing the temperature in 0.4°C increments starting
at 65°C for 85 cycles of 10 sec each. The presence of a single PCR product
was verified both by the presence of a single melting temperature peak
representing a specific product (vs. a nonspecific primer-dimer peak)
using iCycler iQ analysis software and by detection of a single band of
the expected size on a 4.5% Super AcrylAgarose gel.
Real-time RT-PCR was performed in duplicate or triplicate reactions.
Each GSP pair was used with each reaction mix on each of the two different
cDNA templates (derived from macula or periphery). Real-time RT-PCR reactions
for detection of the endogenous control gene, ACTB, were always run in
parallel for each cDNA template in each experimental run as a reference
for accuracy of sample dilution (even if not shown in figure).
Results and Discussion
Experiment 1: Performance Over Time of Mix A Protocol Optimized for Real-Time
RT-PCR
Reactions were carried out using the GSPs that amplified a 128 bp fragment
of the macula-enriched transcript of interest. The amplification curve
for the macula-derived sample crossed a threshold of 100 relative fluorescence
units (RFU) after 21.5 cycles, and the periphery-derived sample crossed
this threshold 1.7 cycles later at 23.2 cycles. These results confirmed
that the transcript of interest was, indeed, enriched in the macula compared
to the rest of the retina (Figure 1A). When the experiment was repeated
one month later using the same reaction components, only the ACTB-derived
PCR products were generated; none of the 128 bp target was detected (data
not shown). The same set of real-time RT-PCR reactions was prepared again
with previously unopened aliquots of each reagent stored at –20°C in a
constant-temperature freezer. Again, only the ACTB-derived transcripts
were amplified (Figure 1B). Traces for late amplifications (CT > 34) of
the 128 bp primer set represent primer-dimers and not specific product,
as determined by melt-curve and gel analysis (not shown). These results
showed that failure to amplify was not due to freeze-thaw induced deterioration
of the stock reagents over time and suggested that some component of the
stock reagents was unstable over time, even at –20°C.
Experiment 2: Comparison of Reactions Based on iQ Supermix vs. Mix A
Use of iQ supermix rescued the assay, resulting in accumulation of the
macula-derived 128 bp products crossing the threshold of 100 RFU after
19.7 cycles (Figure 2) — almost 2 cycles earlier than in the mix A-based
reactions with macula cDNAs for template (Figure 1). In the iQ supermix
reactions containing periphery-derived cDNAs, the 128 bp product crossed
this threshold value at 21.2 cycles (Figure 2), 1.5 cycles later than
the macula reactions with the iQ supermix and almost 2 cycles earlier
than with the mix A periphery reactions in experiment 1 (Figure 1). Equivalent
mix A reactions run at the same time failed to amplify (Figure 2). As
shown in the following experiment, the reproducibility of these results
as well as the stability of the iQ supermix reagents held up over time.
Experiment 3: Performance of iQ Supermix Reactions Over Time
Real-time RT-PCR was performed as described for experiment 2, except that
the iQ supermix (2x) stock used in these reactions had been stored for
4 months at –20°C. The 128 bp segment of the macula-enriched transcript
was again successfully amplified. The amplification curve for the macula
sample crossed the threshold of 100 RFU after 20.1 cycles, while the periphery
sample crossed the threshold 1.7 cycles later at 21.8 (Figure 3). The
iQ supermix is therefore more stable over time than mix A.
Experiment 4: Amplification of a Specific Transcript Using Three Different
Pairs of GSPs With iQ Supermix vs. Mix A
In order to test whether primer design can affect the reproducibility
of amplification curves obtained for a specific transcript in a specific
tissue, real-time RT-PCR was performed using three pairs of primers designed
to amplify different regions of the same target transcript. The three
GSP pairs generate 128 bp, 100 bp, and 99 bp products. Duplicate reactions
using each primer pair with each reaction mix (A or iQ supermix) were
run for each template. The performance of the iQ supermix reactions was
quite consistent for each primer pair (Figure 4A), whereas the performance
of the mix A-based reactions varied for each GSP (Figure 4B).
In the iQ supermix reactions, the average CT for all six traces representing
the amplification of the macula sample with three different primer pairs
was 19.9 ± 0.3 cycles. The average CT for the periphery-derived sample
was 21.4 ± 0.2 cycles, resulting in an average of a 1.5 cycle difference
between the two regions of human neural retina (Figure 4A). This differential
expression profile for the macula-enriched gene transcript was the same
as that obtained in the two previous experiments with iQ supermix reactions
(Figures 2 and 3).
The traces representing the accumulation of PCR products in the mix A-based
reactions varied with each GSP (Figure 4B), in contrast to the traces
for the iQ supermix reactions (Figure 4A). In the mix A-based reactions,
the 128 bp fragment was not amplified at all, whereas the products generated
by the other two primer pairs were amplified at different rates. The amplification
curves for the 100 bp fragment crossed threshold fluorescence at 25.4
cycles in the macula reactions and 25.8 in the periphery, while the curves
for the 99 bp fragment from closer to the 3' end of the target mRNA crossed
the threshold at 21.5 cycles in the macula reactions and 23.1 in the periphery.
The average CT values for the two GSP pairs that resulted in the expectedsized
PCR products were then 23.4 ± 1.9 for the macula and 24.4 ± 1.3 for the
periphery, for an average 1.0 cycle difference (Figure 4B). Clearly, the
real-time RT-PCR results generated using the iQ supermix were more reliable
and reproducible.
Conclusions
Although quantitative real-time RT-PCR is a powerful, sensitive, and reproducible
method to quantitate differences in mRNA expression, many aspects of the
reactions (i.e., primer design, annealing temperatures, and master mix
reagents) must be optimized to put this powerful technology to work successfully
in a new experimental system. Realtime RT-PCR is especially sensitive
to product length, where longer length products and low- to medium-abundance
transcripts cannot be amplified in reactions containing unstable reagents.
While Bio-Rad’s bulletin 2593 (Boeckman et al. 2001) for the iCycler thermal
cycler recommends amplification of PCR products only within the narrow
range of 75–150 bp, we have consistently been able to amplify products
in excess of 300 bp using iQ supermix (data not shown). Replicate CT values
for amplification of the control gene ACTB showed less variation between
replicates and between experiments with either reaction mix (Figures 1
and 3). This was not simply due to the relative abundance of ACTB transcripts
in the samples since the amount of the macula-enriched target gene was
the same as ACTB in the macula. Instead, these results suggest that sequence-related
secondary structure or transcript stability of the target gene could affect
outcomes in the mix A reactions but were not a factor in the iQ supermix
reactions. Finally, the iQ supermix reactions were not only more robust
but also were extremely reproducible: macula-enriched target transcript
CT in the macula-derived samples was 19.9 ± 0.2 cycles (n = 11), and 21.4
± 0.3 cycles (n = 11) in the periphery-derived samples (compare Figures
2, 3, and 4A). Here we have demonstrated that the use of iQ supermix to
optimize reaction conditions allows the best consistency and reproducibility
from experiment to experiment.
Acknowledgements
We are greatly indebted to Drs Gregory J Riggins and Kathy Boon, Duke
University Medical Center, Durham, NC, for their helpful expert advice.
Supported in part by NEI R01 EY 11286, NEI P30 EY 05722 and a Career Development
Award from Research to Prevent Blindness (CBR).
References
Boeckman F et al., Real-time PCR: General considerations, Bio-Rad bulletin
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Bracete AM et al., Isolation of total RNA from small quantities of tissue
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Loging WT et al., Identifying potential tumor markers and antigens by
database mining and rapid expression screening, Genome Res 10, 1393–1402
(2000)
Morrison TB et al., Quantification of low-copy transcripts by continuous
SYBR Green I monitoring during amplification, Biotechniques 24, 954–962
(1998)
The polymerase chain reaction (PCR) process is covered by patents owned
by Hoffman-LaRoche. Use of the PCR process requires a license.
SYBR is a trademark of Molecular Probes, Inc.
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