Hotstart Probe One-Step qRT-PCR Kit
$160.00 - $600.00
$750.00
All products have special prices for bulk purchase, please contact for more details if required.
Cat. No.: HSPQRU-100 (for 100T)
Cat. No.: HSPQRU-500 (for 500T)
Description
Hotstart Probe One-Step qRT-PCR Kit is a high-quality premix based on probes like TaqMan, used for one-step reverse transcription real-time fluorescence quantitative PCR, also known as qRT-PCR (Quantitative Reverse Transcription PCR) or real-time RT-PCR. It is mainly used for highly specific and ultra-sensitive quantitative detection of RNA. Since the probes used are generally TaqMan probes, this method is also often called the TaqMan probe method.
Hotstart Probe One-Step qRT-PCR Kit uses extracted RNA as a template and carries out reverse transcription and fluorescence quantitative PCR in the same reaction tube using qPCR primers, simplifying and speeding up the process, minimizing human error, effectively reducing contamination risk, saving time in PCR experiments, and allowing for high detection throughput.
This product integrates efficient Hotstart M-MuLV Reverse Transcriptase, RNase Inhibitor, and high-quality antibody-bound hot-start Hotstart Taq DNA Polymerase, and optimizes the buffer system, providing superior reverse transcription performance, high detection sensitivity, strong amplification specificity, and good reaction stability. It is highly suitable for detecting low-abundance endogenous RNA, exogenous viral RNA, and other trace RNAs.
This product includes all the general components such as Hotstart M-MuLV Reverse Transcriptase, Hotstart Taq DNA Polymerase, PCR Buffer, dNTPs, stabilizers, Nuclease-free Water, ROX (chosen according to the specific fluorescence quantitative PCR instrument), and magnesium ions, making it easier to operate and more convenient to use. Users only need to prepare primers, probes, and sample RNA.
This product offers Low ROX and High ROX options, making it widely compatible with fluorescence quantitative PCR instruments that do not require ROX or need Low ROX or High ROX as reference dyes. ROX is used to correct fluorescence fluctuations unrelated to PCR, minimizing well-to-well differences. These differences may be caused by various factors, such as pipetting errors and sample evaporation. Different fluorescence quantitative PCR instruments have different requirements for ROX. Please choose High ROX, Low ROX, or no ROX accordingly when preparing the reaction system.
Detection Principle
Probe-based qPCR does not use fluorescent dyes such as SYBR Green to stain PCR products but employs DNA probes labeled with fluorescent groups and quencher groups to target the sequence intended for PCR detection (the designed probe binding sites are usually between the two primer binding sites). Normally, the quencher group on the probe quenches the fluorescent group due to fluorescence resonance energy transfer (FRET). During the PCR reaction, primers and probes anneal to the target gene, and as the primer extends, the 5'→3' exonuclease activity of Taq polymerase degrades the probe bound to the target sequence from the 5' end. When the fluorescent group and the quencher group are separated by Taq polymerase, the quencher group loses its effect, and the fluorescent group can be excited by light to produce fluorescence. Each PCR cycle releases more fluorescent groups, and the fluorescence intensity is proportional to the amount of newly synthesized target fragments, thus achieving quantitative detection. The probe is usually a linear DNA specific to the target sequence, labeled with a fluorescent group such as FAM or HEX at the 5' end and a quencher group such as BHQ1, TAMRA, or MGB at the 3' end.
Features
- Specificity and Sensitivity: The specificity of this product does not solely rely on PCR primers but also on the specificity of the probe. The specific binding and degradation of the probe and the target gene generate fluorescent signals, and the detection sensitivity and specificity are usually significantly higher than methods using fluorescent dyes such as SYBR Green.
- 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. With appropriate optimization of primers and probes, this product can simultaneously detect 2-3 genes.
- High-Quality Hot-Start Enzyme: The Hotstart Taq DNA Polymerase used in this product is a high-quality hot-start enzyme combined with antibodies, enabling convenient and efficient hot-start. The Taq polymerase in Hotstart Taq DNA Polymerase binds to monoclonal antibodies against Taq polymerase, inhibiting its DNA polymerase activity, effectively preventing non-specific amplification caused by non-specific annealing of primers and template DNA or primer dimers at low temperatures. During the pre-denaturation step of the PCR reaction, the antibody is heat-inactivated, ensuring that Taq polymerase activity is only released after pre-denaturation, preventing DNA polymerization before pre-denaturation and greatly improving the specificity, sensitivity, and accuracy of quantitative PCR detection.
Storage
Store at -20℃ in a dark place, effective for two years. Avoid repeated freeze-thaw cycles as much as possible.
Precautions
- The fluorescent labeling of the probe should be determined according to the fluorescence compatibility of the qPCR instrument used. For fluorescence quantitative PCR instruments requiring ROX as a reference dye, avoid using ROX-labeled probes.
- When used for multiplex detection, optimize primers and probes appropriately and use probes labeled with suitable different fluorescent groups. Verify the effect before performing multiplex detection, generally not exceeding triple detection.
- Be careful to avoid RNase contamination by using RNase-free tips, centrifuge tubes, etc.
- Probe One-Step Enzyme Mix (10X) contains a high concentration of glycerol and is highly viscous. Briefly centrifuge to the bottom of the tube before use and gently mix with a pipette, avoiding bubbles as much as possible, then slowly and accurately pipette.
- Ensure that the Probe One-Step Reaction Buffer (2X) is completely thawed before use and mix gently by inverting up and down.
- If the amplification fragments are long or the RNA structure is complex, pre-treat the template RNA at 65℃ for 5-10 minutes to improve reverse transcription efficiency.
- This reaction uses the reverse primer of qPCR as the gene-specific primer for reverse transcription; Random Hexamer Primer or Oligo(dT) Primer commonly used for cDNA first strand synthesis cannot be used.
- Pay attention to the primer annealing temperature. When the annealing temperature is below 60℃, it is recommended to use a three-step PCR amplification method.
- For amplification fragments exceeding 350bp or with high GC content, it is recommended to increase the extension time to 60 seconds or use a three-step method to improve amplification efficiency.
- Tests have shown that repeated freeze-thaw cycles up to 10 times do not significantly affect the use of this product. However, repeated freeze-thaw cycles should still be avoided as they may degrade the product's performance.
- qPCR detection is highly sensitive. The PCR reaction setup area should be kept as free from potential contaminants of the amplifiable products as possible. Seal and discard PCR products to avoid contamination of the experimental environment with high-concentration PCR products.
- This product is for scientific research use by professionals only, not for clinical diagnosis or treatment, not for use in food or medicine, and not to be stored in ordinary households.
- For your safety and health, please wear a lab coat and disposable gloves during operation.
Related:
- Universal Probe One-Step RT-qPCR Kit
- 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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