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.
- Rapid and highly sensitive
- Tolerant of target sequence diversity
- Tolerant of PCR inhibitors
- Utilization of dUTP
- Multiplex detection
- One-step qRT-PCR
- Compatible real-time PCR cycler
|ABI PRISM 7000||Roche
|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|
Store at -20℃, protected from light
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.
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).
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