Description

THUNDERBIRD™ Probe One-step qRT-PCR Kit is a one-step real-time reverse-transcription polymerase chain reaction (RT-PCR) kit using the highly efficient reverse transcriptase "ReverTra Ace™" and Tth DNA polymerase as a PCR enzyme. This product can be used mainly in TaqMan™ probe assays. The one-step system is suitable for high-throughput analysis because of its simple reaction setup. In addition, this system can reduce the risk of cross-contamination. The combination of the two enzymes and optimized buffer system enable the effective detection and quantification of a small amount of RNA. This kit can also detect various kinds of RNA with different sequences because it is tolerant of target sequence diversity.

This product is sold in U.S. under the license of US patent 7772383
from Chakrabarti Advanced Technology NewCo LLC.

Features

• Rapid and highly sensitive
• Tolerant of target sequence diversity
• Tolerant of PCR inhibitors
• Utilization of dUTP
• Multiplex detection

Application

・One-step qRT-PCR
・Compatible real-time PCR cycler

Specification

Table 1.Compatible real-time instruments

Manufacturer	Instrument	Manufacturer	Instrument
Applied Biosystems	ABI PRISM 7000	Roche Diagnostics	LightCycler 2.0
Applied Biosystems	ABI PRISM 7700	Roche Diagnostics	LightCycler Nano
Applied Biosystems	Applied Biosystems 7300	Roche Diagnostics	LightCycler 96/480
Applied Biosystems	Applied Biosystems 7500/Fast	Bio-Rad / MJ	CFX96 Touch
Applied Biosystems	Applied Biosystems 7900HT	Agilent Technologies	Mx3000P/3005P/4000
Applied Biosystems	Applied Biosystems StepOne	TaKaRa	Thermal Cycler Dice
Applied Biosystems	Applied Biosystems StepOneOlus	Qiagen	Rotor-Gene

Category	Item	Details
Storage condition	-	Store at -20℃, protected from light

Components

This kit includes the following components for 250 reactions, with 20 µL per reaction. All reagents should be stored at −20°C.

Item	Details
2× Reaction Buffer*¹	2 × 1.25 mL
DNA Polymerase	125 µL
RT Enzyme Mix	125 µL
50× ROX Reference dye	100 µL
RNase free water	2 × 1.25 mL

*¹2× Reaction Buffer contains essential components for the reaction (buffer, salts, dATP, dCTP, dGTP, and dUTP).

Uracil-N-glycosylase is not supplied with this kit.

Application Data

Example 1. The maximum sensitivities of various one-step qRT-PCR kits

A 4n dilution series of various viral RNAs was detected. The primers and TaqMan™ probes were synthesized in accordance with previous reports. The graph indicates the minimum copy numbers that were detected by the kits. THUNDERBIRD™ Probe One-step qRT-PCR Kit was the only kit that detected all viral RNAs tested at high sensitivity (≤30 copies).

Example 2. Comparison of sensitivity of detection of enterovirus RNA

The sensitivity and quantitativity of various kits were compared by detecting serially (4n ) diluted enterovirus RNA . The primers and probe were synthesized in accordance with a previous report. Applied Biosystems™ StepOnePlusTM was used in this experiment. THUNDERBIRD™ Probe One-step qRT-PCR Kit was the only kit that detected less than 10 copies of RNA and showed wide-ranging quantitation. The results indicate that this kit is suitable for the highly sensitive detection of RNA viruses or mRNA expressed at a low level.

Example 3. Quantification of gene expression using multiplex real-time PCR

The expression levels of IL-1β, TNF-α and GAPDH mRNAs were analyzed using 10n times serially diluted total RNA (1 pg–100 ng) by triplex detection systems with TaqMan™ probes labeled by different fluorescent dyes (Fig.1). LightCycler™ 96 (Roche Diagnostics) was used in this experiment. HeLa S3 cells were incubated for 20 h after being seeded in six-well plates at 4 × 105 cells/well and treated with or without 100 nM phorbol 12-myristate 13-acetate. Then, the expression levels of mRNA were analyzed using purified total RNA from treated cells. The elevations of IL-1β and TNF-α mRNAs were observed upon adding phorbol 12-myristate 13-acetate (Fig. 2).

No significant differences of PCR efficiency and correlation coefficient were observed between the triplex and singleplex.
systems (data not shown).

Fig.1 Multiplex detection

Fig.2 Results of expression analysis using multiplex detection

References

Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl. Environ. Microbiol., 63: 4504–10 (1997)
Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J. Mol. Biol., 306: 469–77 (2001)
Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:762-8 (1999)

Documents

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THUNDERBIRD Probe One-step qRT-PCR Kit-Toyobo