RNAflash™ DCA 1-step RT qPCR (SYBR Green) kit-MAX
Hands-free RNA, a powerful tool for high-throughput qPCR assays
One-click copy of information
Instruction manualPrice:
¥ 6000
Item Number:
500-111
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Specification
- 100T pyrolysis + 300T qPCR
隐藏域元素占位
- Product Description
- Composition
- Precautions
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Product Storage:
Store at -15°C to -25°C for one year; transport with dry ice.
Product Overview:
RNAflash DCA (Direct Cellular Amplification) one-step RT qPCR (SYBR Green) kit is a rapid, highly efficient, all-in-one reagent kit that enables direct conversion from “cells” to “amplification results,” suitable for 10–10 5 Gene expression analysis in cultured cells. This product eliminates the cumbersome, conventional extraction steps, enabling direct genomic DNA removal and RNA isolation directly within cell culture plates. Paired with a one‑step dye‑based RT‑qPCR reagent that delivers high efficiency, robust stability, and exceptional specificity, gene expression results can be obtained from cells in as little as 52 minutes. The kit is easy to use, compatible with a wide range of cell types—including adherent and suspension cells—and accommodates varying cell numbers, enhancing throughput while minimizing sample loss during purification. It also addresses the poor performance often observed with traditional extraction methods when working with low‑cell‑number samples. This kit is suitable for high‑throughput expression analysis of cellular RNA on 96‑ or 384‑well plates.
Product Features:
• Versatility: Gene expression analysis across multiple cell types, enabling efficient detection from as few as 10 to as many as 10⁵ conventional cultured cells or rare cells;
• Purification-free process: Simplifies the conventional RNA extraction workflow, enabling cell lysis within the culture well in 5 minutes and gDNA digestion in under 7 minutes, thereby minimizing RNA loss associated with purification.
• High-throughput compatibility: Meets the demand for parallel processing of large-scale cell samples, significantly reducing both time costs and operational errors in high-throughput assays.
• High efficiency: The optimized one-step RT qPCR system is compatible with lysis buffers, enabling detection in as little as 30 minutes.
Bring your own materials and reagents.
ROX reference dye, 1× PBS buffer, RNase-free ddH2O, RNase-free pipette tips, 1.5 mL RNase-free centrifuge tubes, 0.2 mL RNase-free 8‑strip/PCR tubes, and more.
Experimental Workflow Diagram
Experimental Procedure
RNA acquisition
Sample Processing and Preparation
l Adherent cells:
(a) Cell washing: After gently removing the culture medium from adherent cells in different wells, slowly add an equal volume of pre‑cooled 1× PBS to wash the cells. Centrifuge at 1,000 rpm (930 × g) for 5 min at 4°C to pellet the cells at the bottom of the tube, then carefully discard the PBS and keep the cells on ice for later use.
Note 1: When removing the culture medium or PBS, proceed as gently as possible to avoid cell loss during the washing process.
Note 2: If adherent cells are cultured in 96‑well or 384‑well plates, after removing the culture medium, simply wash the cells with pre‑cooled PBS. If the adherent cells are cultured in other vessels, dissociate them from the vessel walls using trypsin, then inactivate the enzyme by adding an appropriate volume of serum‑containing complete culture medium to neutralize it. After centrifuging to remove the supernatant, wash the cells 2–3 times consecutively with pre‑cooled PBS.
(b) Diluted cells: According to experimental requirements (the linear range for cell number testing is 10 to 10 5 (1), dilute the cells with pre-cooled 1× PBS, and aliquot the diluted cell suspension into 200 μL PCR tubes, 5 μL per tube (maintaining a cell count of 10–10). 5 per tube), and keep on ice for later use.
l Suspension cells:
(a) Remove the culture medium: Transfer the suspended cells to a centrifuge tube, centrifuge at 1000 rpm (930 × g) for 5 min at 4°C to pellet the cells at the bottom of the tube, and discard the supernatant.
(b) Cell washing: Add pre-cooled 1× PBS (ensure the cell density is approximately 500 μl/10). 4 -10 6 (Per cell), gently pipette up and down 8–10 times until no visible cell clumps remain; centrifuge at 1,000 rpm (930 × g) for 5 minutes at 4°C to pellet the cells, then discard the PBS.
(c) Cell dilution: According to experimental requirements (the linear range for cell count testing is 10 to 10 5 (1), dilute the cells with pre-cooled 1× PBS, and aliquot the diluted cell suspension into 200 μL PCR tubes, 5 μL per tube (maintaining a cell count of 10–10). 5 per tube), and keep on ice for later use.
Note: When removing the culture medium or PBS, proceed with utmost care to avoid cell loss during the washing process.
RNA acquisition
(a) Prepare the lysis working solution (in a 200 μL PCR tube)
Component Volume (μL) Lyse™ Buffer 48 Purge™ Enzyme 2 Note: Prepare the lysis working solution according to the volume required for each well plate, with a component ratio of Lyse™ Buffer : DNase I : Purge™ Enzyme = 24 : 1. After preparation, invert and mix 10–15 times, avoiding vigorous vortexing; once mixed, place on ice and use within 1 hour.
(b) Add 50 μL of lysis working solution to each well of the 96-well plate containing cells, gently pipette up and down 8–10 times to ensure thorough mixing, and incubate at room temperature for 5 minutes to lyse the cells.
(c) Add 5 μL of StopXtract™ Buffer, gently pipette up and down 8–10 times to mix, and let stand at room temperature for 2 minutes to terminate the reaction.
Note: The cleavage products can be stored on ice for 2 hours; for long-term storage, they should be kept at −80°C.
One-step RT-qPCR reaction
Configure the reaction system
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) DCA One-Step RT-qPCR Enzyme Mix 1 DCA One-Step RT-qPCR Buffer Mix 10 10 µM target upstream primers (2, 3, 4) 0.4 10 µM downstream primer for the target sequence 0.4 10 µM target probe 0.2 Cell lysis buffer 2 (Recommended) ROX Reference Dye (5) X RNase-Free/DNase-Free Water Up to 20 μL Note: 1. Thaw each component on ice, gently mix and centrifuge, then add the reagents in order from highest to lowest concentration.
2. In general, a final primer concentration of 0.2 μM in the reaction mixture yields good amplification. When the reaction performance is suboptimal, the primer concentration can be adjusted within the range of 0.1–1.0 μM.
3. In general, a final probe concentration of 0.1 μM in the reaction system yields good amplification results. When the reaction performance is suboptimal, the probe concentration can be adjusted within the range of 0.05–0.25 μM.
4. Please select an amplicon length within the 100–500 bp range; a length of 100–200 bp is particularly recommended. You may increase the number of targets as needed for your assay.
5. If using a qPCR instrument that requires dye-based normalization, add the dye to the reaction mixture at the concentration recommended in the precautions.
Template Deployment
Add 2 μL of cell lysis buffer to each PCR reaction tube in the experimental group, and use 2 μL of RNase‑Free/Nuclease‑Free H₂O as the no‑template control (NTC) in each group; each group includes three replicates.
Note: A recommended volume of 2 μL of cell lysis buffer is suggested, as it does not inhibit amplification. Avoid using less than 1 μL to minimize errors caused by inaccurate pipetting.
Reaction procedure
Gently mix the reaction mixture, briefly centrifuge, and then run the protocol outlined in Table 2.
Table 2 Reaction Protocol for the One-Step RT-qPCR (Probe-Based) Kit
Steps Number of cycles Reaction temperature Reaction time Collect signals Reverse transcription (5) 1 cycle 50℃ 5 min No Pre-degeneration 1 cycle 95℃ 20 sec No Transgender 40 cycle
95℃ 3 sec No Extension 60℃ 30 sec Is Note: 5. 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.
No RT Control response (optional)
No RT Control refers to a reverse transcription–negative control reaction performed without reverse transcriptase, used to verify the absence of genomic DNA contamination in the RNA template. Probe qPCR Mix can also be employed for this assay.
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 Storage conditions 500-104-1 Lyse™ Buffer (a) 2500 µL per vial Lysis buffer Dissolve and store at 2–8°C. 500-104-2 Purge™ Enzyme 100 µL per vial Genome-removing enzyme -20℃ 500-104-3 StopXtract™ Buffer (b) 250 µL per vial Termination buffer Store the aliquots at −20°C. 500-104-4 DCA One-Step RT-qPCR Enzyme Mix 100 µL per vial Enzyme mixture -20℃ 500-104-5 DCA One-Step RT-qPCR Buffer Mix(c) 1000 µL per vial Buffer mixture -20℃ a. Lyse™ Buffer remains stable for one year when stored at 2–8°C after thawing.
b. StopXtract™ Buffer should be protected from repeated freeze-thaw cycles and stored in aliquots at −20°C.
c. DCA One-Step RT-qPCR Buffer Mix contains a dNTP mix, Mg²⁺, hot-start Taq polymerase, reverse transcriptase, and other components; after opening the package, this component must be stored at low temperature. Product Composition:
Item number Component Specification (100T/20 μL) Explanation Storage conditions 500-104-1 Lyse™ Buffer (a) 2500 µL per vial Lysis buffer Dissolve and store at 2–8°C. 500-104-2 Purge™ Enzyme 100 µL per vial Genome-removing enzyme -20℃ 500-104-3 StopXtract™ Buffer (b) 250 µL per vial Termination buffer Store the aliquots at −20°C. 500-104-4 DCA One-Step RT-qPCR Enzyme Mix 100 µL per vial Enzyme mixture -20℃ 500-104-5 DCA One-Step RT-qPCR Buffer Mix(c) 1000 µL per vial Buffer mixture -20℃ a. Lyse™ Buffer remains stable for one year when stored at 2–8°C after thawing.
b. StopXtract™ Buffer should be protected from repeated freeze-thaw cycles and stored in aliquots at −20°C.
c. DCA One-Step RT-qPCR Buffer Mix contains a dNTP mix, Mg²⁺, hot-start Taq polymerase, reverse transcriptase, and other components; after opening the package, this component must be stored at low temperature.
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Precautions:
1. Store reagents according to the specified storage conditions and requirements; for example, to avoid repeated freeze-thaw cycles of StopXtract™ Buffer, aliquot it and store at −20°C.
2. When lysing cells, pipette gently to avoid excessive foaming.
3. The one-step RT-qPCR (probe-based) enzyme reaction solution contains a high concentration of glycerol; before use, briefly centrifuge to collect the liquid at the bottom of the reaction tube, and then pipette.
Gently pipette to ensure thorough mixing, then accurately aspirate.
4. When preparing the reaction mixture, use RNase-free pipette tips, Eppendorf tubes, and other consumables to minimize the risk of contamination.
5. This product does not contain ROX Reference Dye. If ROX Reference Dye needs to be added to the reaction to correct inter‑well fluorescence signal variability,
Please determine, according to the instrument’s user manual, whether to add ROX Reference Dye. (Typically, instruments requiring high‑concentration ROX correction use a working concentration of 0.4 μM, while those requiring low‑concentration ROX use 0.08 μM; see Table 3 for instrument‑specific details.) It is recommended to dilute the 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.
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