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: firstname.lastname@example.org
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.
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 l 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 g/reaction) using oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers instructions.
Gene-specific primers (GSPs) were designed to anneal near the 3' end of two mRNA transcripts and to generate PCR products 75300 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 l of template cDNA per 20 l 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 l of a 1:1,500 dilution of 10,000x SYBR Green I stock, 500 nM of each GSP, and 1 l of cDNA. iQ supermix reactions consisted of iQ supermix (Bio-Rad) at a final concentration of 1x, 10 nM fluorescein calibration dye, 1 l of a 1:1,500 dilution of 10,000x SYBR Green I stock, 500 nM of each GSP, and 1 l of cDNA. To control for pipetting losses, 19 l of each 20 l reaction was amplified in a 96- well thin-wall PCR plate (Bio-Rad) using the following PCR parameters: 95C for 2 min followed by 50 cycles of 95C for 15 sec, 60C for 15 sec, and 72C for 15 sec. Melt-curve analysis was performed immediately following amplification by increasing the temperature in 0.4C increments starting at 65C 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 20C 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 20C.
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 20C. 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.
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-Rads bulletin 2593 (Boeckman et al. 2001) for the iCycler thermal cycler recommends amplification of PCR products only within the narrow range of 75150 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.
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).
Boeckman F et al., Real-time PCR: General considerations, Bio-Rad bulletin 2593 (2001)
Bracete AM et al., Isolation of total RNA from small quantities of tissue and cells, Focus 21, 3839 (1999)
Loging WT et al., Identifying potential tumor markers and antigens by database mining and rapid expression screening, Genome Res 10, 13931402 (2000)
Morrison TB et al., Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification, Biotechniques 24, 954962 (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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