Quantification of Zea mays mRNAs by Real-Time PCR Using the Universal ProbeLibrary

The Universal ProbeLibrary (UPL) is a detection system for real-time PCR based on locked nucleic acid (LNA) probes. We tested the efficiency of the UPL for the quantification of maize transcripts on two detection platforms. The results indicate that the UPL is an attractive alternative to assays based on intercalating dyes or conventional probes.

Introduction

Quantitative real-time PCR (qPCR) is an important method to measure gene expression levels, and thus to obtain information about gene function in vivo. In general, there are two different methods of qPCR assays either based on intercalating dyes (e.g., SYBR Green I) or using a sequence-specific detection probe. In dye-based assays, the intercalating dye binds and detects non-specifically any double-stranded DNA that accumulates during the PCR like by-products and primer-dimers. In probe-based assays, a labeled sequence-specific probe ­anneals to the amplification product between the primer bind­ing sites. Therefore, only the correct amplicon contain­ing both primer and probe binding sites will generate a signal. Until now, the disadvantage of probe-based assays has been that they are less flexible than dye-based assays. The Universal ProbeLibrary has been designed to eliminate this disadvantage. The li-brary includes 165 different probes, each made up of only 8–9 nucleotides. The probes contain modified ”locked“ nucleic acids [1] and therefore efficiently anneal to their binding site at standard annealing temperatures despite their short length. On the other hand, the small probe size allows probe binding to several positions in the transcriptome, and different mRNAs can thus be detected by one probe. Specificity of the assays is warranted by the combination of primers and probes. The Universal ProbeLibrary is either available as a complete probe set or organism specif-ic sets for human, rat, and mouse. Intron-spanning assays can be designed using the online Assay Design Center (www.universalprobe­library.com) for human, rat, mouse, Drosophila, C. elegans, Arabidopsis, primates, and recently also for maize and zebrafish.

Here, we evaluated the quality of the Universal ProbeLibrary for Zea mays by comparing newly designed Universal ProbeLibrary assays and established SYBR Green I assays. Additionally, differences in performance between two quantitative real-time PCR platforms, the LightCycler® Carousel-based System and the ABI PRISM® 7000 Sequence Detection System, were investigated.

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Materials and Methods

RNA isolation and reverse transcription

RNA was prepared from 100 mg ground leaf material following the TRIZOL-bromo-chlorpropane protocol [2]. To remove traces of contaminating DNA, 1 U of DNase I per µg RNA and MgCl2 to a final concentration of 2 mM were added and reactions were incubated for 15 minutes at 37°C, followed by a denaturation step of 15 minutes at 70°C. cDNA synthesis was performed with approximately 1 µg of total RNA and 50 pmol of random nonamer primers.

Quantitative PCR

qPCR with SYBR Green I detection with the LightCycler® Instrument was performed using the LightCycler® FastStart DNA MasterPLUS SYBR Green I, whereas the FastStart SYBR Green I Master (Rox) was used for SYBR Green I assays on the ABI PRISM® 7000 Sequence Detection System. Quantification via Universal ProbeLibrary was performed with the LightCycler® TaqMan® Master and the FastStart TaqMan® Probe Master (Rox). SYBR Green I assays were performed with 300 nM primers and Universal ProbeLibrary assays with 200 nM primers and 100 nM probe (Table 1). All other reaction conditions were as described by the manufacturer.

Amplification conditions using the ABI PRISM® 7000 Sequence Detection System were 10 minutes of initial denaturation at 95°C, followed by 40 cycles of each 15 seconds at 95°C and 1 minute at 60°C. Amplification conditions with the LightCycler® Instrument were 10 minutes of initial denaturation at 95°C, followed by 45 cycles of each 20 seconds at 95°C, 20 seconds at 60°C, 20 seconds at 72°C for SYBR Green I assays, and 10 minutes of initial denaturation at 95°C, followed by 45 cycles of each 10 seconds at 95°C, 30 seconds at 60°C, and 1 second at 72°C for the UPL assays, respectively. For all SYBR Green I assays, a melting curve from 60°C to 95°C was recorded.

Results and Discussion

We designed Universal ProbeLibrary assays for 20 differ-ent maize (Zea mays) mRNA sequences as a representative subset indicating whether the Universal ProbeLibrary is suitable for the detection of maize transcripts. Half of the tested RNAs encode enzymes of the photosynthetic machinery and are therefore highly expressed in green leaf tissues. The remaining RNAs were randomly chosen from maize RNA sequences present in the databases. For comparison of the performance of the two different detection principles, optimized SYBR Green I assays were available [3].

Sequence input into ProbeFinder software at the online Assay Design Center (www.universalprobelibrary.com) was straightforward, and information about the designed assays was clearly laid out.

All assays were run on a sequential 1:4 dilution series of maize cDNA. Fifteen of 20 designed assays showed excellent performance without the need for optimization. The standard curves deduced from the dilution series showed a perfect slope over the expected range of dilutions based on the expression level. For three of the five failed assays, products were amplified according to gel analysis, but not detected by the probe. For the remaining two assays, no amplification was detectable. This might also be due to absence of expression in the tested tissue.

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The success rate is lower compared with Universal ProbeLibrary assays designed for other organisms where 95%–99% is achieved. This result may be explained by the fact that the maize genome is not yet complete-ly sequenced, and probes and primers may bind to unknown sequence elements in uncharacterized transcripts.

In general, crossing points of Universal ProbeLibrary assays occured slightly later, but sensitivity and specific-ity were similar to SYBR Green I assays. The main advantage is exemplarily shown in Figure 1: With both assay systems, the abundant photosynthetic phosphoenolpyruvate carboxylase (PEPC) cDNA was amplified from the six leaf cDNA dilutions tested. However, the negative con-trol showed an amplification signal in the SYBR Green I assay, but not in the Universal ProbeLibrary assay. Melting curve analysis revealed that the amplification signal in the negative control of the SYBR Green I assay resulted from primer-dimers that were not detected by the Universal ProbeLibrary probe. This was confirmed by gel electrophoresis (data not shown).

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We additionally compared the performance of the Universal ProbeLibrary on two different qPCR platforms, the LightCycler® Instrument and the ABI PRISM® 7000 Sequence Detection System. Figure 2 shows a representative result with a primer-probe combination specific for phosphoenolpyruvate carboxykinase (PEPCK), a photosynthetic enzyme of intermediate abundance. Universal ProbeLibrary assays worked efficiently on both systems. The crossing points were usually slightly lower with the LightCycler® Instrument, and this occasionally resulted in a higher sensitivity.

Conclusions

The Universal ProbeLibrary together with the Assay De-sign Center provides a simple, but efficient alternative for the quantitative detection of transcripts from maize mRNA.

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References

1. Braasch DA, Corey DR (2001) Chem Biol 8:1–7

2. Chomczynski P, Mackey K (1995) Anal Biochem 225:163–164

3. Hahnen S et al. (2003) Photosynthesis Res 75:183–192

This article was originally published in Biochemica 1/2007, pages 8-10. ©Springer Medizin Verlag 2007