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Cat. No.: PQRU-100 (for 100T)

Cat. No.: PQRU-500 (for 500T)

Description

Probe One-Step qRT-PCR Kit (20X, UDG) is a new high-quality carryover prevention kit for one-step reverse transcription real-time quantitative PCR based on probes such as TaqMan. It's primarily used for the ultra-sensitive quantitative detection of RNA. This product contains an optimized ratio of high-quality UDG enzyme and dUTP, effectively eliminating false positives or lowered CT values caused by product contamination during PCR amplification. Since the commonly used probe is TaqMan, this method is often referred to as the carryover prevention TaqMan probe method.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between the uracil (dU) base in the DNA strand containing uracil and deoxyribose, releasing free uracil. It is primarily used to eliminate product contamination issues during PCR amplification. Its contamination prevention principle involves adding an appropriate amount of dUTP in the PCR reaction to substitute dTTP into the DNA, producing PCR amplification products with dU bases. In subsequent PCR reactions, UDG enzyme selectively cleaves any single or double-stranded DNA containing dU brought in by potential contamination from previous PCR amplifications, preventing the contamination from negatively impacting the current PCR amplification.

The Probe One-Step qRT-PCR Kit (UDG) uses extracted RNA as a template. It conducts reverse transcription and fluorescence quantitative PCR consecutively in the same reaction tube. The process is simple and quick, minimizes manual errors, effectively reduces contamination risks, saves PCR operation time, and has a large detection throughput.

This product integrates the highly efficient M-MuLV Reverse Transcriptase, RNase Inhibitor, and the superior antibody-bound BeyoFast™ Taq DNA Polymerase. The buffer system has been optimized for excellent reverse transcription performance, high detection sensitivity, strong amplification specificity, and stable reactions. It's particularly suitable for detecting low abundance endogenous RNA, exogenous viral RNA, and other minute RNA samples.

Principle

The probe-based qPCR does not use fluorescent dyes like SYBR Green to stain PCR products. Instead, it uses a DNA probe labeled with a fluorescent group and a quencher targeting the sequence intended for PCR detection (the designed probe binding site is typically between the two primer binding sites). Normally, the quencher on the probe causes fluorescence quenching of the fluorescent group due to Förster resonance energy transfer (FRET). During PCR reaction, both the primer and probe anneal to the target gene. As the primer extends, the 5'→3' exonuclease activity of Taq enzyme causes the probe bound to the target sequence to degrade from the 5' end. After the Taq enzyme cuts the probe's fluorescent group and quencher, the quencher's effect disappears, allowing the fluorescent group to produce fluorescence when excited. Each PCR cycle releases more fluorescent groups, with fluorescence intensity directly proportional to the number of newly synthesized target fragments, enabling quantitative detection. The probe is typically a linear DNA specific to the target sequence, with its 5' end labeled with a fluorescent group like FAM or HEX, and its 3' end labeled with a fluorescence quenching group like BHQ1, TAMRA, or MGB.

Features

• High specificity and sensitivity: Specificity isn't solely reliant on PCR primers. Only when the probe specifically binds and degrades with the target gene can a fluorescent signal be generated. Its detection sensitivity and specificity are typically significantly higher than methods using fluorescent dyes like SYBR Green.
• For multiplex detection: In a single reaction well, different genes correspond to different probes, and different probes correspond to different fluorescent labels, enabling multiplex fluorescence quantitative PCR detection. Testing shows that, after properly optimizing primers and probes, this product can be used simultaneously to detect 2-3 genes.
• Hotstart property: The Hotstart Taq DNA Polymerase used in this product is a high-quality heat-activated enzyme bound with antibodies. It allows convenient and efficient heat activation. In Hotstart Taq DNA Polymerase, the Taq enzyme binds with a monoclonal antibody against Taq enzyme, inhibiting the enzyme's DNA polymerase activity, effectively preventing non-specific annealing of primers and template DNA or primer dimerization at low temperatures. The antibody is deactivated by heating in the pre-denaturation step of the PCR reaction, ensuring the release of Taq enzyme activity only after pre-denaturation, preventing DNA polymerization before then, thereby significantly improving PCR reaction specificity, sensitivity, and quantitative detection accuracy.
• Easy to use: This product contains M-MuLV Reverse Transcriptase, Hotstart Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, stabilizers, Nuclease-free Water, ROX (selected based on different fluorescence quantitative PCR machines), magnesium ions, and all other universal components, making operations simpler and use more convenient. Users only need to provide primers, probes, and sample RNA.

ROX Dye

This product offers both Low ROX and High ROX, making it broadly compatible with fluorescent quantitative PCR machines that either don't require ROX or require Low ROX or High ROX as a reference dye. The role of ROX is to correct fluorescence fluctuations unrelated to the PCR process, thereby minimizing inter-well differences. These variations can be caused by several factors, such as pipetting errors and sample evaporation. Different fluorescent quantitative PCR machines have different requirements for ROX. Depending on the specific equipment in use, one should choose either a high concentration ROX (High ROX), low concentration ROX (Low ROX), or no ROX Probe when setting up the reaction system.

Storage

Store in the dark at -20ºC. Valid for two years. Avoid repeated freeze-thaw cycles.

Precautions

• The fluorescent labeling of the probe must be determined based on the fluorescence compatibility of the qPCR instrument being used. For fluorescent quantitative PCR machines that require ROX as a reference dye, avoid probes labeled with ROX.
• When used for multiplex detection, it's essential to appropriately optimize primers and probes and use probes labeled with suitable distinct fluorescent groups. Before conducting multiplex detection, confirm its effectiveness; it's generally recommended not to exceed triple detection. If using for triple or quadruple detection, we recommend Multiplex Probe qPCR Mix (2X, UDG).
• Please avoid RNase contamination and use RNase-free pipette tips, centrifuge tubes, etc.
• Probe One-Step Enzyme Mix (20X, UDG) contains a high concentration of glycerol and is viscous. Before use, briefly centrifuge to collect the contents at the bottom of the tube, then gently mix with a pipettor. Try to avoid introducing air bubbles during the mixing process and then pipette slowly and accurately.
• Ensure that the Probe One-Step Reaction Buffer (2X) is fully thawed before use. Invert and mix gently before application.
• If the amplification fragment is long or the RNA structure is complex, pre-treat the template RNA at 65ºC for 5-10 minutes to improve reverse transcription efficiency.
• For this reaction, the qPCR's Reverse Primer serves as the gene-specific primer for reverse transcription. Do not use Random Hexamer Primer or Oligo(dT) Primer, which are typically used for cDNA first-strand synthesis.
• Take note of the primer annealing temperature. When the annealing temperature is <60ºC, it's recommended to use a three-step PCR amplification method.
• For amplicons exceeding 350bp or with a high GC content, consider extending the elongation time to 60 seconds or use the three-step method to improve amplification efficiency.
• Tests show that repeated freeze-thawing up to 10 times doesn't significantly impact the product's performance. Nevertheless, try to minimize freeze-thawing, as this might degrade the product over time.
• qPCR detection is ultra-sensitive. Even though this product has excellent anti-contamination properties, the area where the PCR reaction is set up should still be kept free from potential contamination. Dispose of PCR products securely to prevent contamination of the experimental environment by high-concentration PCR products.
• This product is intended for scientific research use by professionals only. It must not be used for clinical diagnosis or treatment, nor for food or drugs, and should not be stored in ordinary residences.
• For your safety and health, please wear lab coats and disposable gloves when handling.

Related:

• Probe One-Step qRT-PCR Kit (20X, UDG)
• Multiplex Probe qPCR Mix (2X, UDG)
• Probe qPCR Mix (2X, UDG)
• SYBR Green One-Step qRT-PCR Kit (20X, UDG)
• SYBR Green qPCR Mix (2X, UDG)

Only for research and not intended for treatment of humans or animals

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