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Brilliant Core Reagent Kits for QPCR and QRT-PCR



Economical quantitative PCR (QPCR) reagent kits for use with all types of fluorescent probes

Gothami Padmabandu Lisa Grismer Reinhold Mueller
Stratagene

Stratagenes Brilliant quantitative PCR and RT-PCR core reagent kits are compatible with many fluorescent detection technologies. Several versions have been created to optionally include dUTP/uracil-N-glycosylase (UNG) decontamination control reagents. All kits include SureStartTaq DNA polymerase , which provides the benefits of hot-start PCR, greatly reducing nonspecific primer annealing and extension that can lead to amplification of undesirable products. We evaluated the Brilliant quantitative PCR and RT-PCR core reagent kits in a variety of experiments, including the amplification of DNA and RNA targets followed by the detection of amplicon with various fluorescent chemistries. In this article we describe the data generated by Brilliant core reagents which include SureStart Taq and dUTP/UNG decontamination control.

Quantitative PCR has become the technology of choice for investigating gene expression and gene copy number. One growing application is the validation of differential expression signals seen with microarrays. While microarrays can reveal expression level changes, the current level of noise in microarray technology often warrants a secondary validation of expression level changes. Similarly gene copy number measurements are subject to significant experimental error. New quantitative PCR methods rely on real-time PCR analyses and generate a fluorescence value that indicates the amount of product amplified to that point in the reaction.1,2 In contrast to endpoint analysis, real-time quantitation is based on measurements taken during the exponential phase of amplification, a time when PCR efficiency is least influenced by reaction variables. Multiplex QPCR is the ultimate answer to precise quantitation. Measuring several amplicons simultaneously in the same reaction vessel gives the most precise QPCR and QRT-PCR results. This article describes an open-system family of enzyme, buffer, and reference dye reagent kits that can be used with most QPCR and QRT-PCR probe systems.

Real-Time Assessments with Versatile Options

Kit Name

Enzyme

dUTP/UNG

Passive Reference Dye

Number of reactions

U.S. List Price

U.S. List Price / reaction

Catalog

Brilliant QPCR

SureStart Taq

No

Separate Tube

200

$150

$0.75

#600530

Brilliant Plus QPCR

SureStart Taq

Yes

Separate Tube

200

$225

$1.13

#600540

TaqMan PCR Core Reagent Kit

AmpliTaq Gold

Yes

In Buffer

200

$375

$1.88

#N808-0228

Platinum Quantitative PCR SuperMix-UDG

Platinum Taq

Yes

No

500

$625

$1.25

#11730025

Brilliant core reagent kits support quantitative amplification and detection with multiplex capability and show consistent high performance with various fluorescent detection systems, including molecular beacons, TaqMan probes, single-dye primers, and Scorpions probes.3 Table 1 lists the various kits and their components. The QPCR kits are used with DNA templates, either to detect DNA mutations or to measure gene copy number. The QRT-PCR kits are used with RNA templates, typically for measuring RNA expression levels. Mutations can also be detected in expressed RNA with these kits (Table 2).

Kit Name

Enzyme

dUTP/UNG

Passive Reference Dye

Number of reactions

U.S. List Price

U.S. List Price/ reaction

Catalog #

Brilliant Single-Step QRT-PCR

SureStart Taq and StrataScript Reverse Transcriptase

No

Separate Tube

200

$225

$1.13

#600532

Brilliant Two-Step QRT-PCR

SureStart Taq and StrataScript Reverse Transcriptase

No

Separate Tube

200

$400

$2.00

#600534

Brilliant Plus Single-Step QRT-PCR

SureStart Taq and StrataScript Reverse Transcriptase

dUTP only

(UNG not included in single-step kit

Separate Tube

200

$250

$1.25

#600542

Brilliant Plus Two-Step QRT-PCR

SureStart Taq and StrataScript Reverse Transcriptase

Yes

Separate Tube

200

$475

$2.38

#600544

TaqMan Gold RT-PCR Core Reagent Kit

AmpliTaq Gold and MultiScribe RT

dUTP only for single-tube kit

In Buffer

200

$700

$3.50

#N808-0232

Platinum Quantitative RT-PCR ThermoScript
One-Step System

Platinum Taq ThermoScript

No

No

500

$1,575

$3.00

#11721023

Available in either a single- or two-step format (for those who wish to archive a portion of the RT reaction prior to PCR), the Brilliant and Brilliant Plus QRT-PCR kits include high-performance StrataScript RNaseH-minus reverse transcriptase and SureStart Taq DNA polymerase, which has a hot-start feature. For compatibility with carry-over contamination-control strategies, the Brilliant Plus kits also include dUTP and uracil-N-glycosylase (UNG) for decontamination of amplicons. (The Single-Step RT-PCR Kit contains dUTP only.) While the dUTP provides a convenient decontamination strategy, incorporation of dUTP is slightly less efficient than dTTP and the Brilliant Plus kits are slightly less sensitive than the standard Brilliant kits.

By including a passive reference dye as a separate component, reaction volume pipeting error can be normalized from well to well. Users have the option of including or not including the passive reference dye in their reactions. While a passive reference dye is very helpful, it does consume some of the useable light spectrum and thus reduces the number of different dyes that can be used in muliplex reactions.

Comparison Tests Confirm High Sensitivity

Fig.1
We compared Stratagenes Brilliant Plus quantitative PCR core reagent kit with another commercially available core reagent kit to amplify and detect a 294-bp region in exon 3 of the human b-actin gene using a TaqMan probe (Figure 1). Three concentrations (100, 1, and 0.01 ng) of human genomic DNA were amplified (Methods), and both tests included dUTP and UNG (Methods), identical primers and linear hydrolysis probe, and a passive reference dye. Our data show that for each concentration of target DNA, the threshold cycle (Ct; the cycle at which fluorescence is first detected above background) was comparable or better for the Brilliant Plus kit. For 100, 1, or 0.01 ng of human genomic DNA, the Ct values generated with Brilliant Plus reagents were 18.0, 25.6, or 33.0, respectively; the corresponding Ct values for reactions with the other commercially available kit were 18.3, 26.0, or 34.3 (Figure 1).

To demonstrate the sensitivity and absolute quantification capabilities of Stratagenes Brilliant Plus QPCR core reagent kit, we used molecular beacon chemistry to amplify and detect a 148-bp fragment from the gene encoding the mouse muscle nicotinic acetylcholine receptor g-subunit. In this experiment, the passive reference dye was not included. We achieved a detection level of 101 to 108 copies of plasmid DNA (Figure 2) with our standard protocol (Methods). When the Ct was plotted versus the input amount of target, the resulting standard curve was linear over eight orders of magnitude, with an R2 value of 0.998 (Figure 2). This large dynamic range eliminated the need to analyze serial dilutions of experimental samples for gene quantification. Moreover, this target titration demonstrated that the Brilliant Plus QPCR kit is sensitive enough to detect as few as 10 copies of plasmid DNA.

To test the compatibility of the Brilliant Plus QPCR core reagent kit with different fluorescent chemistries, we used a Scorpions probe to detect and amplify the 134-bp rpoB gene target of Mycobacterium tuberculosis, an emergent pathogen. We amplified and detected 100, 1000, and 10,000 copies of the M. tuberculosis target gene; the respective Ct values were 38.2, 34.9, and 31.6 (data not shown).

Multiplex Capabilities

Fig.3

In further experiments, we showed that the Brilliant Plus QPCR reagent kits can efficiently detect more than one dye per reaction (Multiplex QPCR or MxQPCR). We used Stratagenes molecular beacon SDF-1 (Stromal Derived Factor 1) Allelic Discrimination kit, which detects a common G-to-A substitution that may delay or accelerate the onset of AIDS in humans (Figure 3). We detected this substitution by amplifying human genomic DNA in the presence of two molecular beacons in the same tube, each molecular beacon with a different dye (FAM and TET). In this scheme, the FAM probe was complementary to the mutant allele (A), and the TET probe was complementary to the wild-type allele (G). We included the passive reference dye, although not required, as the third fluorophore. The genotype-specific control DNA samples supplied in the molecular beacon SDF-1 kit were used for this allelic discrimination.

This multiplex assay provides a way of determining whether a DNA sample is derived from a homozygous wild-type, heterozygous, or homozygous mutant individual. One way of calling alleles is by comparing Ct values. In a sample containing the wild-type alleles only, the TET signal should be relatively strong and should appear in earlier amplification cycles (Ct = 26), whereas the FAM signal should be at background level (Ct = 40; Figure 3 ). In the case of a heterozygote, both FAM and TET signals should be seen, but the Ct values should be slightly higher than that for the homozygote wild type or mutant because only one allele each (i.e., the wild-type and the mutant allele) is detected. The Ct values for the heterozygote were 27 (TET) and 27 (FAM) (Figure 3).

Another method for calling alleles is to examine the fluorescence at the last annealing stepin this case, at cycle 40. The final fluorescence provides a qualitative readout because components in the reactions might be limiting at the end of PCR. However, when the proper dilutions of target DNA are included in the reaction, the final fluorescence is often related to the amount of the wild-type or mutant allele in the sample (cycle 40; Figure 3) and can be easily extrapolated by the software of the real-time reader (Table 3). In this analysis, the wild-type sample had a TET signal of 2.2 (normalized endpoint fluorescence), the mutant sample had a FAM signal of 1.34, and the heterozygote showed approximately one-half of the TET or the FAM signal.

FAM (mutant)

TET (wild type)

G/G (wild type)

0.02

2.20

A/A (mutant)

1.34

0.02

G/A (heterozygote)

0.88

1.23

Brilliant QRT-PCR in Single- and Two-Step Formats

The reverse transcription reaction is the first critical step of RT-PCR procedures. To ensure that this reaction is optimized, Stratagene has included StrataScript Rnase H-minus RT in both the single-step and two-step formats of the Brilliant and Brilliant Plus QRT-PCR core reagent kits. StrataScript reverse transcriptase is a genetically modified Moloney murine leukemia virus reverse transcriptase with a point mutation that results in removal of RNase H activity. In numerous tests, StrataScript reverse transcriptase has produced high yields of cDNA and long transcripts, providing ideal cDNA templates for subsequent PCR amplifications.4

The Brilliant single-step QRT-PCR core reagent kits include all the necessary reagents for cDNA synthesis and PCR amplification in a single tube. This format uses primers specific to the RNA of interest. Although UNG cannot be used in the single-step format, the Brilliant Plus kit includes dUTP in the nucleotide mix. Should any amplicon be released from the single-step RT-PCR, the decontamination control would then eliminate amplification of contaminating DNA in subsequent PCR or two-step RT-PCR.

The Brilliant two-step QRT-PCR core reagent kit is ideal for use when sensitivity of the reverse transcription reaction needs to be increased. This increased sensitivity is achieved through the provided buffers that are specifically optimized for the first-strand synthesis and for the PCR amplification. With the two-step format, which includes both random oligomers and oligo(dT) primers, only a small portion of the reverse transcription reaction is used for PCR amplification. Consequently, cDNA samples can be archived or used for amplifying multiple targets. With the Brilliant Plus two-step QRT-PCR format, UNG can be used for controlling carry-over contamination.

Compatibility with Fluorescent Chemistries

Total RNA

Threshold Cycle (Ct)

100 ng

14.8

10 ng

18.8

1.0 ng

22.1

100 pg

25.6

10 pg

28.5

1.0 pg

32.3

0.1 pg

35.2

We tested the Brilliant single-step QRT-PCR core reagent kits with a number of fluorescent probes to confirm the kits compatibility with different fluorescent chemistries. We used a linear hybridization probe specific for detecting human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA with the Brilliant single-step QRT-PCR core reagents. The indicated amounts of human total RNA were added. Table 4 shows the real-time analysis of this target titration for the abundant GAPDH mRNA. As expected, both the no-reverse transcriptase (no-RT) control and the no-template control did not show any amplification (Figure 4).

To evaluate performance, we compared Stratagenes Brilliant single-step QRT-PCR kit to a another commercially available quantitative RT-PCR one-step system by amplifying and detecting a 150-bp region of human GAPDH mRNA using the Molecular Beacon GAPDH Expression Analysis kit. For this comparison, we amplified three concentrations (100, 1, and 0.01 ng) of human total RNA (Methods); however, because the other commercially available kit did not provide a passive reference dye, none was included in this experiment. We found that for each concentration of target RNA, the Ct was comparable or better for the Brilliant kit (data not shown). We assessed the sensitivity of the Brilliant two-step QRT-PCR kit to detect a low-abundance target by amplifying and detecting hypoxanthine phosphoribosyl transferase (HPRT) mRNA from human brain total RNA. For the real-time analysis of the RT-PCR amplification, we used the primers and linear hydrolysis probe from the TaqMan pre-developed assay reagents (PE Biosystems) in the Brilliant Plus two-step QRT-PCR kit. We included UNG and the passive reference dye.The following Ct values were obtained: 22.5 (100 ng), 26.4 (10 ng), 29.1 (1 ng), 32.7 (100 pg), and 37.7 (10 pg) (data not shown). The results show that with the Brilliant Plus two-step quantitative RT-PCR kit, a low-abundance mRNA can be detected from as little as 10 pg of total RNA.

Conclusions

The Brilliant QPCR and QRT-PCR core reagent kits provide highly sensitive and versatile quantitative assessments for real-time gene quantification and gene expression analysis. The kits are compatible with linear hydrolysis (TaqMan probes), molecular beacon, single dye primers, and Scorpions fluorescent technologies. With the Brilliant QPCR core reagent kit, we detected as few as 10 copies of mouse muscle nicotinic acetylcholine receptor g-subunit target. For research parameters that favor convenience, speed, high-throughput capabilities, and reduced risk of sample contamination, the QRT-PCR single-step format is the method of choice. When the highest sensitivity is required, the Brilliant two-step RT-PCR core format is ideal. The Brilliant core reagent kits can be used with various fluorescent chemistries, with or without the passive reference dye, and with UNG decontamination protocols. Furthermore, they compare favorably with other commercially available kits.

Methods

QPCR: A typical 50-l reaction contained the following reagents: 1X Brilliant Plus QPCR core buffer; the optimal concentration of MgCl2 (3.0 to 5.5 mM); sequence-specific primers (300 to 600 nM); probe (200 to 400 nM molecular beacon, 200 to 400 nM TaqMan probe, and 400 nM Scorpions probe); 200 M each dGTP, dATP, dCTP, and 400 M dUTP; 0.5 units of uracil-N-glycosylase ; 2.5 U of SureStart Taq DNA polymerase; and target nucleic acid template (genomic DNA or plasmid DNA as indicated in the Figure legends). Each experiment included control reactions that lacked template-"no-template" controls. All reactions were performed in duplicate. Thermal cycling conditions are as specified for two-step RT-PCR (see below). Other commercially available core reagents or master mixes, TaqMan, and Scorpions chemistries were tested according to the recommendations of each respective manufacturer. The M. tuberculosis rpoB Scorpions and PCR primers are a kind gift of Dr. David Whitcombe of Astra-Zeneca.

Single-Step QRT-PCR: A typical RNA amplification reaction with either molecular beacons or a linear hydrolysis probe contained the following components: 1X Brilliant Plus QRT-PCR buffer 3.0; the optimal concentration of MgCl2 (4.0 to 5.5 mM); 200 M each of dGTP, dATP, dCTP, and 400 M of dUTP; optimal concentration of probe (200 to 400 nM molecular beacon or linear hydrolysis probe); 10 U of StrataScript reverse transcriptase (RT); 2.5 U of SureStart Taq DNA polymerase; and RNA template (total RNA, poly (A)+ RNA, or in vitro transcribed RNA). Each experiment was performed with no-RT controls, in which StrataScript RT was omitted from the reaction and with no-template controls. Other commercially available reagents and TaqMan chemistry were tested according to the manufacturers recommendations. The following thermal cycling conditions were used for testing molecular beacons with the Brilliant single-step quantitative RT-PCR core kit: 45C for 30 minutes; 95C for 10 minutes; 40 cycles of 95C for 30 seconds, 55C for 1 minute; followed by 72C for 30 seconds. The following thermal cycling conditions were used for testing linear hybridization probes with the Brilliant single-step quantitative RT-PCR core kit: 45C for 30 minutes; 95C for 10 minutes; 40 cycles of 95C for 30 seconds; followed by 60C for 1 minute. All RT-PCR reactions can be cycled in a temperature cycler designed to detect changes in fluorescence in real time.

Two-step QRT-PCR: All first-strand synthesis reactions were performed in 200-l thin-wall reaction tubes in a 10-l reaction volume with Stratagenes RoboCycler 96 temperature cycler. The RT reaction contained 1X RT buffer, 5.5 mM MgCl2, 1 mM each dNTP, reverse primer, 100 ng of random oligomers, and RNA template. Reactions in which the RT was omitted were included in each experiment. The reactions were annealed at 25C for 10 minutes [for oligo(dT) or random oligomers], after which 10 U of StrataScript RT was added. The reactions were incubated at 45C for 30 minutes, then at 95C for 5 minutes. An aliquot (1 l) of the first-strand synthesis reaction was added to optical tubes containing 1X Brilliant Plus QPCR core buffer; the optimal concentration of MgCl2; 200 M each of dGTP, dATP, dCTP, and 400 M of dUTP; 0.5 units of UNG; optimal concentrations of sequence-specific primers and probe; and 2.5 U of SureStart Taq DNA polymerase in a final volume of 50 l. A no-template control was included. The PCR cycling conditions for molecular beacons are as follows: 50C for 2 minutes; 95C for 30 seconds; 55C for 1 minute; and 72C for 30 seconds. The thermal cycling conditions were 50C for 2 minutes; 95C for 10 minutes; for monitoring TaqMan reactions, this was followed by 40 cycles of 95C for 15 seconds and 60C for 1 minute. A temperature cycler designed to detect changes in fluorescence in real time was used.

REFERENCES
  1. Higuchi, R., et al. (1992) Bio/Technology 10: 413-417.

  2. Higuchi, R., et al. (1993) Bio/Technology 11: 1026-1030.

  3. Whitcombe, D. et al (1999) Nature Biotechnol. 17: 804-807.

  4. Nielson, K. et al (1993) Strategies 6: 45-46


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