RNAflash™ DCA 1-step RT qPCR (SYBR Green) kit
Hands-free RNA, a powerful tool for high-throughput qPCR assays
One-click copy of information
Instruction manualPrice:
¥ 4000
Item Number:
500-103
-
Specification
- 50T lysis + 100T qPCR
隐藏域元素占位
- Product Description
- Composition
- Precautions
-
Item number: 500-103
1. Product Storage:
- Store at -15°C to -25°C for one year; transport using dry ice.
2. Product Overview:
RNAflash DCA (Direct Cellular Amplification) one‑step RT qPCR (SYBR Green) kit is a rapid, highly efficient, end‑to‑end reagent system that enables direct conversion of “cells” into “amplification results,” suitable for gene expression analysis in cultures ranging from 10 to 10^5 cells. This product eliminates the cumbersome steps of conventional extraction, allowing genomic DNA removal and RNA isolation directly within cell culture plates. Paired with a highly efficient, stable, and specific one‑step dye‑based RT‑qPCR reagent, it delivers gene expression data 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 and addressing the poor performance often observed with traditional extraction methods at low cell densities. It is also well suited for high‑throughput analysis of cellular RNA on 96‑ or 384‑well plates.
3. Product Features:
Universality: Gene expression analysis across multiple cell types, enabling robust detection from as few as 10 to as many as 10⁵ conventional cultured cells or rare cells.
Purification-free process: Streamlines the conventional RNA extraction workflow, achieving cell lysis within 5 minutes directly in the culture well and completing genomic DNA (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 52 minutes.
4. Product Composition: (50T Pyrolysis + 100T qPCR)
Item number Component Specification (50T+100T/20 μL) Explanation Storage conditions 500-103-1 Lyse™ Buffer (a) 2500 µL per vial Lysis buffer Dissolve and store at 2–8°C. 500-103-2 Purge™ Enzyme 100 µL per vial Genome-removing enzyme -20℃ 500-103-3 StopXtract™ Buffer (b) 250 µL per vial Termination buffer Store the aliquots at −20°C. 500-103-4 DCA One-Step RT-qPCR Enzyme Mix 100 µL per vial Enzyme mixture -20℃ 500-103-5 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 a fluorescent dye; after opening, this component should be stored away from light.
5. Bring Your Own Materials and ReagentsROX 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.
6. Experimental Workflow Diagram
7. Experimental Procedure
7.1 RNA Extraction
7.1.1 Sample Handling and Preparation
l Adherent cells:
(a) Cell washing: After gently removing the culture medium from adherent cells in different wells, add an appropriate volume of trypsin to each well and incubate at 37°C for 3–5 minutes. Observe cell morphology under a microscope. When cells begin to round up and intercellular spaces widen, digestion is nearly complete. Add an adequate volume of serum‑containing culture medium to stop the digestion, then gently pipette the cells to detach them and form a single‑cell suspension. Centrifuge at 1,000 rpm (930 × g) for 5 minutes at 4°C. Discard the supernatant, wash once with an equal volume of pre‑cooled 1× PBS, and centrifuge again at 1,000 rpm (930 × g) for 5 minutes at 4°C to pellet the cells. Resuspend the cells in an appropriate volume of PBS (the linear range for cell counting is 10 to 10^5 cells), and keep on ice until further use.
Note 1: When removing the culture medium or PBS, proceed with utmost care 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 adherent cells are cultured in other vessels, dissociate them from the vessel wall 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) Cell dilution: According to experimental requirements (the linear range for cell quantification is 10 to 10^5 cells), dilute the cells with pre‑cooled 1× PBS. Aliquot the diluted cell suspension into 200 μL PCR tubes, dispensing 5 μL per tube (maintaining a cell concentration of 10–10^5 cells per tube), and keep on ice until further 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 (aiming to maintain a cell density of 2 × 10^4 to 2 × 10^6 cells/mL, at 500 μL per 10^5 cells), and 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 quantification is 10 to 10^5 cells), dilute the cells with pre‑cooled 1× PBS. Aliquot the diluted cell suspension into 200 μL PCR tubes, dispensing 5 μL per tube (to maintain a cell concentration of 10–10^5 cells per tube), and keep on ice until further use.
Note: When removing the culture medium or PBS, proceed with utmost care to avoid cell loss during the washing process.
7.1.2 RNA Extraction
(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, and gently pipette up and down 8–10 times to ensure thorough mixing. 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.
7.2 One-step RT-qPCR reaction
7.2.1 Setting up the Reaction System
Prepare the reaction mixture on ice according to Table 1.
Table 1 Reaction System for One-Step RT-qPCR (Fluorescent Dye-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 upstream primers for the target (2,3,4) 0.4 10 µM downstream primer for the target sequence 0.4 Cell lysis buffer X (refer to step 7.2.2) ROX Reference Dye (5) Y 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 descending order of volume.
2. In general, a final primer concentration of 0.2 μM in the reaction mixture yields good amplification results. When the reaction performance is suboptimal, the primer concentration can be adjusted within the range of 0.1–1.0 μM.
3. The number of targets can be increased as needed for testing.
4. Please select an amplicon length within the 100–500 bp range; a length of 100–200 bp is particularly recommended.
5. If using a qPCR instrument that requires dye-based normalization, add the dye to the reaction mixture at the concentrations recommended in the precautions.
7.2.2 Formwork Deployment
Add 2–8 μL of cell lysate to each PCR reaction tube in the experimental group, and use 5 μL of RNase‑Free/Nuclease‑Free H₂O as a no‑template control (NTC) sample; each group includes three replicates.
Note: Adding 2–8 μL of cell lysis buffer does not inhibit amplification. Ensure that the volume of cell lysis buffer added is no less than 1 μL to avoid errors caused by inaccurate pipetting.
7.2.3 Reaction Procedure
Gently mix the reaction mixture, briefly centrifuge, and then run the protocol described in Table 2.
Table 2 Reaction Protocol for One-Step RT-qPCR (Fluorescent Dye-Based) Kit
Step Cycle Number Reaction Temperature Reaction Time Signal Acquisition Reverse transcription (5) 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 Melting (6) Select according to the instrument model. Note: 5. For templates with complex secondary structures or high GC‑rich 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.
6. Typically, after the extension step, a melting step is required to assess amplification specificity; please select the appropriate protocol based on your instrument model.
7.2.4 No RT Control Response (Optional)
No RT Control refers to a reverse transcription–negative control reaction performed without reverse transcriptase, used to determine whether genomic DNA is present in the RNA template. This assay can also be conducted using the Fast Taq SYBR Green qPCR Mix (Cat. No. 500-102).
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 (fluorescent dye–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 variation,
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 correction use 0.08 μM; see Table 3 for specific instrument 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.
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;
-
Product Composition: (50T Pyrolysis + 100T qPCR)
Item number Component Specification (50T+100T/20 μL) Explanation Storage conditions 500-103-1 Lyse™ Buffer (a) 2500 µL per vial Lysis buffer Dissolve and store at 2–8°C. 500-103-2 Purge™ Enzyme 100 µL per vial Genome-removal enzyme -20℃ 500-103-3 StopXtract™ Buffer (b) 250 µL per vial Termination buffer Store the aliquots at −20°C. 500-103-4 DCA One-Step RT-qPCR Enzyme Mix 100 µL per vial Enzyme mixture -20℃ 500-103-5 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 a fluorescent dye; after opening, this component should be stored away from light.
-
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 (fluorescent dye–based) enzyme reaction solution contains a high concentration of glycerol; before use, briefly centrifuge to collect the solution 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 variation,
Please determine, according to the instrument’s user manual, whether to add ROX Reference Dye. Generally, instruments requiring high‑concentration ROX correction use a working concentration of 0.4 μM, while those requiring low‑concentration ROX correction use 0.08 μM; see Table 3 for specific instrument details. For the ROX Reference Dye product, a 50× dilution is recommended 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.
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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