RNAflash™ DCA 2-step RT qPCR (probe) kit
Hands-free RNA, a powerful tool for high-throughput qPCR assays
One-click copy of information
Instruction manualPrice:
¥ 4000
Item Number:
500-106
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Specification
- 50T lysis + 100T 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.
Product Overview
RNAflash DCA (Direct Cellular Amplification) Two‑Step RT qPCR (Probe) Kit is a rapid, highly efficient, end‑to‑end reagent kit that enables direct conversion of “cells” into “amplification results,” suitable for gene expression analysis in 10 to 10⁵ cultured cells. This product eliminates the cumbersome traditional extraction steps, allowing genomic DNA removal and RNA isolation directly within the cell culture plate. Paired with high‑efficiency, highly stable, and highly specific reverse transcription reagents and probe‑based qPCR reagents, it delivers gene expression data from cells in as little as 82 minutes. The kit is easy to use, compatible with a wide range of cell types—including adherent and suspension cells—and adaptable to varying cell numbers, enhancing efficiency while minimizing sample loss during purification and addressing the poor performance of conventional extraction methods when working with low cell counts. It is also well suited for high‑throughput analysis of cellular RNA using 96‑ or 384‑well plates.
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 reverse transcription system is compatible with the lysis buffer, and the qPCR assay enables precise quantification of the resulting cDNA, with a turnaround time as short as 50 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, 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 1000 rpm (930 × g) for 5 minutes at 4°C. After discarding the supernatant, wash once with an equal volume of pre‑cooled 1× PBS, followed by 1000…
Centrifuge at 930 × g and 4°C for 5 minutes to pellet the cells, then resuspend the pellet in an appropriate volume of PBS (the linear range for cell counting is 10 to 10^5 cells). Keep on ice until further 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 2–3 times with pre‑cooled PBS. If the 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 the trypsin. 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 concentration of 10–10). 5 per tube), and keep on ice for later use.
l Suspension cells:
(c) Remove the culture medium: Transfer the suspended cells to a centrifuge tube, centrifuge at 1000 rpm (930 × g) for 5 minutes at 4°C to pellet the cells at the bottom of the tube, and discard the supernatant.
(d) Cell washing: Add pre-cooled 1× PBS (ensure the cell density is maintained at 2 × 10…). 4 Up to 2 ×10 6 cells/mL, with 10³ to 10⁵ cells per 500 μL), 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.
(a) 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 concentration 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, and gently pipette up and down 8–10 times to ensure thorough mixing. Incubate at room temperature for 5–7 minutes to lyse the cells.
(c) Add 5 μL of StopXtract™ Buffer, gently pipette up and down 8–10 times to mix, and incubate 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.
Reverse transcription reaction
Configure the reaction system
Prepare the reaction mixtures on ice according to Table 1, using 5 μL of RNase‑Free/Nuclease‑Free H2O as the no‑template control (NTC) and including three replicates per group. Then perform the reactions following the protocol in Table 2: reverse transcription at 50°C for 20 min, followed by enzyme inactivation at 85°C for 5 min. Store the reaction products on ice if they will be used immediately; otherwise, store them at −20°C for later use.
Table 1 Reverse Transcription Reaction System
Reagent Single reaction volume (μL) (1) Single reaction volume (μL) (1) DCART Enzyme Mix 1.75 3.5 DCART Buffer Mix 3.25 6.5 Cell Lysis Buffer (2) X X RNase-Free/DNase-Free Water Up to 10 μL Up to 20 μL Note: 1. Thaw each component on ice, gently mix and centrifuge, then add the reagents in order of decreasing volume.
2. Typically, add 4 μL of cell lysis buffer to a 20 μL reaction mixture. Adjust the volume according to the gene’s expression level, but do not exceed 8 μL. Ensure that the volume of cell lysis buffer added is no less than 1 μL to minimize errors caused by inaccurate pipetting.
Table 2 Reverse Transcription Reaction Protocol
Steps Number of cycles Reaction temperature Reaction time Reverse transcription (3) 1 cycle 50℃ 20 min Transgender 85℃ 5 min Note: 3. The reverse transcription time may be appropriately extended depending on the initial template quantity or gene abundance, with a maximum reaction time of no more than 30 minutes.
qPCR reaction
Configure the reaction system
Prepare the reaction mixture on ice according to Table 3.
Table 3 DCAqPCR (probe) reaction system
Reagent Single reaction volume (μL) (4) DCAqPCR probe Mix 10 10 µM upstream primers for the target (5, 6, 7) 0.4 10 µM downstream primers for the target (5, 6, 7) 0.4 10 µM target probe (8) 0.2 cDNA template X (refer to step 7.3.2) ROX Reference Dye (9) Y RNase-Free/DNase-Free Water Up to 20 μL Note: 4. Thaw each component on ice, gently mix, and centrifuge; then add the reagents in order of decreasing volume in the reaction mixture.
5. 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.
6. The number of targets can be increased as needed for the assay.
7. Please select an amplicon length within the 100–500 bp range; a length of 100–200 bp is particularly recommended.
8. 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.
9. If using a qPCR instrument that requires dye-based normalization, add the dye to the reaction mixture at the concentrations recommended in the precautions.
Template Deployment
Add 2–4 μL of cell lysis buffer to each PCR reaction tube in the experimental group, and use 5 μL of RNase‑Free/Nuclease‑Free H₂O as the no‑template control (NTC) group; 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 not less than 1 μL to avoid errors caused by inaccurate pipetting.
Reaction procedure
Gently mix the reaction mixture, briefly centrifuge it, and then run the protocol outlined in Table 2.
Table 2 DCAqPCR (probe) reaction protocol
Step Cycle Number Reaction Temperature Reaction Time Signal Acquisition Pre-degeneration 1 cycle 95℃ 30 sec No Transgender 40 cycle
95℃ 10 sec No Extension 60℃ 30 sec is 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. It can also be detected using the DCA qPCR probe Mix (Catalog No. 500-106-6).
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 500-106-1 Lyse™ Buffer(a) 250 µL per vial Lysis buffer Dissolve and store at 2–8°C. 500-106-2 Purge™ Enzyme 100 µL per vial Genome-removing enzyme -20℃ 500-106-3 StopXtract™ Buffer(b) 250 µL per vial Termination buffer Store the aliquots at −20°C. 500-106-4 DCA RT Enzyme Mix(c) 175 µL per vial Enzyme mixture -20℃ 500-106-5 DCA RT buffer Mix(c) 325 µL per vial Buffer mixture -20℃ 500-106-6 DCA qPCR probe Mix(d) 1000 µL per vial Quantitative 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 RT Mix contains a dNTP mix, Mg²⁺, reverse transcriptase, oligo d(T)₁₈, random primers, and cofactors. This component must be stored at the specified temperature, and no additional specific primers are required during reverse transcription.
d. The DCA qPCR probe mix contains hot-start Taq polymerase, UDG, dNTPs, and other components, and must be stored under the specified conditions.
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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 vigorous bubbling.
3. The DCA RT Enzyme Mix contains a high concentration of glycerol; before use, briefly centrifuge the tube to collect the solution at the bottom, then gently pipette up and down to resuspend it.
After thorough mixing, accurately pipette.
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. 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. 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.
Keywords
GOONIE
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