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.

FEATURES

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

APPLICATIONS

  • One-step qRT-PCR
  • Compatible real-time PCR cycler
Applied
Biosystems
ABI PRISM 7000 Roche
Diagnostics
LightCycler 2.0
ABI PRISM 7700 LightCycler Nano
Applied Biosystems 7300 LightCycler 96/480
Applied Biosystems 7500/Fast Rio-Rad/MJ CFX96 Touch
Applied Biosystems 7900HT Agilent Technologies Mx3000P/3005P/4000
Applied Biosystems StepOne TaKaRa Thermal Cycler Dice
Applied Biosystems StepOneOlus Qiagen Rotor-Gene

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.

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.Comparison of sensitivity of detection of enterovirus RNA

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).

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)