Description

THUNDERBIRD™ Next Probe qPCR Mix is a 2x concentration premixed reagent for real-time PCR using various detection systems such as TaqMan™ probes and fluorescently labeled primers. The components other than ROX (provided separately), probes, and primers are pre-mixed, making the preparation of the reaction solution convenient. Additionally, this kit minimizes variation in fluorescence intensity between samples, allowing for highly reproducible results.

This product has further improved the composition of the conventional THUNDERBIRD™ Probe qPCR Mix [Code No. QPS-101], optimizing it for multiplex detection and detection from samples including PCR inhibitors. It is also compatible with high-speed PCR cycles.

Features

• Broad dynamic range
High efficiency and specific amplification enable analysis over a broad dynamic range.
• Fast cycles with 10 s extension times achievable
Targets can be detected even in fast PCR with an extension time of 10s.
• Improved quantitative performance in the presence of PCR inhibitors
Efficient amplification can be performed even in the presence of PCR inhibitors.
• Enhanced multiplex detection performance
Efficient multiplex detection amplification can be performed.
• Enhanced stability of prepared reaction mixtures
Our unique Buffer composition improves the stability of primer-template mixtures.
• Compatibility with dUTP carryover prevention
The use of dUTP prevents false positives due to carryover contamination*.
• Compatibility with various real-time thermocyclers and reverse transcription reagents
This kit can be used with typical real-time PCR instruments, and can be combined with various RT reagents. This kit combined with Toyobo's RT reagents (ReverTra Ace™ series and SuperPrep™ series) has been shown to enable highly accurate and sensitive expression analysis.

* Uracil-DNA Glycosylase (UNG) is not supplied with this kit. Uracil-DNA Glycosylase (UNG), Heat-labile [Code No.UNG-101] , sold separately, is required.

Specification

Category	Item	Details
Storage condition	-	Store at -20℃

Components

This kit includes the following components for 100 reactions (QPX-101T) and 500 reactions (QPX-101), with a total of 20μL per reaction. All reagents should be stored at -20°C.

QPX-101T	Details
THUNDERBIRD™ Next probe qPCR Mix	1mL × 1
50× ROX reference dye	50μL × 1

QPX-101	Details
THUNDERBIRD™ Next probe qPCR Mix	1.67mL × 3
50× ROX reference dye	250μL × 1

Application Data

Example 1.Quantification of GAPDH cDNA (total RNA 0.2 pg ~ 20 ng equivalent)

GAPDH was detected using a fluorescent dye (FAM) labeled TaqMan™ probe.
cDNA from HeLa cell total RNA (total RNA 0.2 pg ~ 20 ng equivalent) was used.
Analyses confirmed high quantitative performance over a broad dynamic range from 0.2 pg to 20 ng.

Example 2.Comparison of normal/fast cycles in multiplex detection system

β-actin, GAPDH, PLA, and TUB genes were detected using a multiplex detection system with TaqMan™ probes labeled with four different fluorescent dyes in a "Normal cycle" with an extension time of 30 s and in a "Fast cycle" with an extension time of 10s.
4-fold dilutions [5 steps] of cDNA derived from HeLa cell total RNA were used as samples and analyzed using Bio-Rad CFX96.
High PCR efficiency was demonstrated in fast cycles, even in multiplex detection system where amplification is difficult.

Example 3.Evaluation of multiplex performance in the presence of inhibitors (EtOH, hematin, heparin, humic acid, UTM, EDTA)

β-actin, GAPDH, PLA, and TUB genes were detected using a multiplex detection system with TaqMan™ probes labeled with four different fluorescent dyes in the presence of the inhibitors described in the figure below.
4-fold dilutions [5 steps] of cDNA from HeLa cell total RNA were used as samples and analyzed using Bio-Rad’s CFX96.
Addition of PCR inhibitors caused amplification failure with other companies' reagents. By contrast, using THUNDERBIRD™ Next Probe qPCR Mix, no significant difference in efficiency was observed even when inhibitors were added.

Example 4.Evaluation of multiplex performance in the presence of inhibitors (feces, whole blood, saliva)

gDNA derived from Shigella, VTEC, and Salmonella were detected using a multiplex detection system with TaqMan™ probes labeled with four different fluorescent dyes. These targets were detected in the presence of PCR inhibitors (feces, whole blood, and saliva). These detections were performed using Bio-Rad's CFX96.
While other companies' reagents cannot efficiently amplify in a multiplex detection system in the presence of PCR inhibitors, THUNDERBIRD™ Next Probe qPCR Mix can detect as low as 10 copies of each bacterial gDNA even in the presence of PCR inhibitors.

Example 5.Stability of prepared reaction mixtures (including primers, probes, and templates)

After mixing primers, probes, and templates in PCR mixtures, target amplification was performed immediately or after incubation for 48 hours at room temperature, protected from light.
Ct value delayed after 48 hours with other companies' products; however, THUNDERBIRD™ Next Probe qPCR Mix showed stable performance even after 48 hours.
If there is a difference in ΔCt of 0.5 or more, or if ΔCt cannot be detected, the column is highlighted in yellow.

Documents

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THUNDERBIRD Next Probe qPCR Mix-Toyobo