1-step RT-qPCR (Probe) kit
One run completes reverse transcription and qPCR, yielding Ct values in 45 minutes.
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Instruction manual- Product Description
- Composition
- Precautions
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Product Storage
Store at -15°C to -25°C for one year.
Product Overview
A one-step RT‑qPCR (probe‑based) kit, specifically designed for quantitative PCR assays using RNA as the template (e.g., RNA viruses). By employing gene‑specific primers (GSP), reverse transcription and PCR are performed in a single tube, eliminating the need for additional open‑tube handling or pipetting. This significantly increases assay throughput while minimizing the risk of contamination. The kit supports rapid single‑tube single‑plex or multiplex fluorescent‑probe‑based RT‑qPCR reactions and effectively prevents cross‑contamination. It includes a high‑performance reverse transcriptase, a hot‑start Taq DNA polymerase, and an optimized buffer system; only primers/probes and RNA are required to initiate the reaction.
Product Features
This product is widely used for the detection of various RNA‑based nucleic acids in animals, plants, and microorganisms (including viruses). 
This product performs reverse transcription and qPCR in a single tube, simplifying the workflow, enhancing efficiency, and effectively reducing the risk of contamination from multiple handling steps. 
A unique enzyme combination ensures high specificity and high sensitivity, with a detection limit as low as 10 fg. 
The optimized buffer system ensures efficient reverse transcription and amplification. Instructions for Use
1. Prepare the reaction mixture on ice according to Table 1.
Table 1 Reaction System for One-Step RT-qPCR (Probe-Based) Kit
Reagent Single reaction volume (μL) (1) One-step RT-qPCR (probe-based) reaction buffer 10 One-step RT-qPCR (probe-based) enzyme reaction solution 1 10 µM upstream primers for the target (2,3,4) 0.4 10 µM downstream primer for the target sequence 0.4 10 µM target probe 0.2 ROX Reference Dye (5) X RNase-Free/DNase-Free Water Up to 15 μL Note: 1. Thaw each component on ice, gently mix, and centrifuge; then add the reagents in order of decreasing volume in the reaction mixture.
2. In general, a final primer concentration of 0.2 μM in the reaction mixture yields good amplification results. When amplification performance is suboptimal, the primer concentration can be adjusted within the range of 0.1–1.0 μM. Additionally, the molar ratio of primers to probes is typically 2:1, though this can be fine-tuned based on the observed performance of the primers and probes.
3. Depending on the assay requirements, an appropriate fluorescence channel can be selected to increase the number of targets.
4. Please select an amplicon length within the 100–500 bp range; a length of 100–200 bp is particularly recommended.
5. (To be provided by the user) If using a qPCR instrument that requires dye-based normalization, add the dye to the reaction mixture at the concentrations recommended in the precautions.
2. Add 5 μL of positive RNA template (10 fg–100 ng) to each PCR reaction tube in the experimental group, and use 5 μL of RNase‑Free/H2O as the no‑template control (NTC group). Each group includes three replicates.
Note: The optimal template input range is 10 pg to 1 ng; excessive input may inhibit amplification, while insufficient input can lead to unstable results.
3. Gently mix the reaction mixture, briefly centrifuge, and then run the protocol described in Table 2.
Table 2 Reaction Protocol for the One-Step RT-qPCR (Probe-Based) Kit
Step Number of cycles Reaction temperature Reaction time Signal acquisition Reverse transcription (6) 1 cycle 50℃ 3 min No Pre-degeneration 1 cycle 95℃ 30 sec No Transgender 40 cycle
95℃ 10 sec No Extension 60℃ 30 sec is Note: 6. For templates with complex secondary structures or high-GC regions, increasing the reverse transcription temperature to 55°C can enhance amplification efficiency and sensitivity. The reverse transcription time may be extended to 15 minutes, which helps increase cDNA yield.
Instrument No need to add ROX.
(Bio-Rad) IQ5, CFX96, CFX384, CFX Connect, MJOpticon, Opticon 2;
(Cepheid) SmartCycler® System, Smart Cycler II System;
(Roche) LightCycler®2.0, 480, 96;
(Qiagen) Rotor-Gene Q, 3000, 6000;
(Bioer) Line-Gene;
(Eppendorf) Mastercycler ep realplex;
(Analytik Jena) qTOWER3;
Add ROX
(Final concentration: 0.4 μM)
(Thermo) ABI7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOnePlus
Add ROX
(Final concentration: 0.08 μM)
(Thermo) ABI 7500, 7500 Fast, ViiA7, QuantStudio 3 / 5, QuantStudio 6 / 7 / 12K
Flex, QuantStudioTMDx;
(Agilent) Mx3000P, Mx3005P, MX4000;
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Product Composition
Item number Component Specification (100T/20 μL) Explanation 500-108-1 One-step RT-qPCR reaction buffer (probe-based) 1000 µL per vial Reaction Buffer Premix 500-108-2 One-step RT-qPCR reaction mix (probe-based) 100 µL per vial Enzyme premix -
Precautions
1. The one-step RT-qPCR (probe-based) enzyme reaction solution contains a high concentration of glycerol. Before use, briefly centrifuge the tube to collect the liquid at the bottom, then gently pipette up and down to ensure thorough mixing before accurately dispensing the volume.
2. When preparing the reaction solution, please use RNase-free pipette tips, Eppendorf tubes, and other consumables to minimize the risk of contamination.
3. This product does not contain ROX Reference Dye. If ROX Reference Dye is required in the reaction to correct inter‑well fluorescence signal variations, please follow the instrument’s user manual to determine whether to add it. Generally, instruments that require high‑concentration ROX calibration use a working concentration of 0.4 μM, while those requiring low‑concentration ROX calibration use 0.08 μM (see Table 3 for specific instrument requirements). For this product, we recommend diluting ROX Reference Dye 50‑fold in the reaction mixture (e.g., add 1 μl of ROX Reference Dye to a 50 μl reaction system). If the experimental results are suboptimal, you may adjust the amount of ROX Reference Dye added.
Keywords
GOONIE
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Goonie Biotechnology (Guangzhou) Co., Ltd.
Room 321–332, Building G5, No. 39 Ruihe Road, Huangpu District, Guangzhou
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