The QStrip One-Step RT-qPCR Mix has been freeze-dried to a bead format. Beads are stable at room temperature and designed to perform a fast and easy methods for one-step reverse transcription real-time PCR (RT-qPCR). The QStrip One-Step RT-qPCR Mix is provided in 0.1 ml tubes containing one lyophilized bead for one reaction. The lyophilized bead contains reverse transcriptase, DNA polymerases, dNTPs and buffer components at optimal concentrations. The additional reagents required are water, primers, and template RNA. The supermix is applicable for TaqMan probes system. The reverse transcriptase is modified from that be able to tolerate high temperature to 61°C, which is helpful for reverse transcription of RNA templates to unravel the complex RNA secondary structures. The specific DNA polymerase allows for high specificity and reproducible amplification.
Storage
Store at room temperature stably up to the expiry date.
Features
➤Lyophilized beads form – stable in room temperature.
➤Premix components to ensure greater reproducibility between reactions,
minimize pipetting steps, and reduce the potential for pipetting errors and contamination.
➤Time efficiency – included most reagents for RT-qPCR reaction.
➤Thermal flexible- the temperature of reverse transcription can be raised to 61°C.
Applications
➤Detection of expressed genes. ➤Examination of transcript variants.
Composition
| Contents | MB309-0008 | MB309-0048 |
|---|---|---|
| QStrip One-Step RT-qPCR Mix | 8 Reactions | 48 Reactions |
Quality Control
Each lot of QStrip One-Step RT-qPCR Mix is functional test to ensure lot-to-lot reproducibility
Required Materials
➤qPCR thermal cycling instrument
➤RNase-free water
➤Gene-specific primers and fluorogenic probe.
➤ROX reference dye (optional)
➤RNase inhibitor (optional)
Compatible Instruments
Depending on the use of fluorescent of PCR instrument, check on the following table, optimal cycling conditions will vary with different instruments:
| Product Name | RT-qPCR Thermal Cycling Instrument |
|---|---|
| QStrip One-Step RT-qPCR Mix | Applied BiosystemsTM StepOne & StepOnePlus Applied BiosystemsTM QuantStudio 3 and 5 |
One-Step RT-qPCR Kit Protocol
1. Confirm the lyophilized beads are in sink to the bottom of the tube before use.
2. Set up the following reaction mixture (compatible with TaqMan probes)
| Component | Volume |
|---|---|
| QStrip One-Step RT-qPCR Mix | 1 Bead |
| Forward Primer (10 μM) | 0.4 μl |
| Reverse Primer (10 μM) | 0.4 μl |
| TaqMan Probe (10 μM) | 0.4 μl |
| ROX reference dye (optimal) | Variable |
| RNA sample (Final to 1 pg ~0.1 μg) | ≤2 μl |
| Add Nuclease-free water to | 20 μl |
Note: Optimal amount of final primer concentration is 0.2 μM. The final primer concentration can be adjusted in the range of 0.1 ~ 0.4 μM. The concentration of probe can be varying depended on he real-time PCR instruments and the probe data sheets. The volumes of RNA samples should be below to ≤1/10 per reaction. The amount of RNA should be 1 pg ~0.1 μg. Too much amounts of RNA may inhibit the RT-qPCR reaction.
3. Add the prepared mixture to the tube containing the lyophilized bead.
Note: Do not directly touch the lyophilized bead with the pipette tips. Please add the supermix along to
the wall of the tubes. After the lyophilized bead is dissolved, mix well until the reagent becomes
clear to ensure the reagent is completely dissolved.
4. Spin down the reaction solution.
5. Perform RT-qPCR reactions using the following cycling program.
*Fluorogenic data should be collected during this step through the reporter channel.
Note: The cycling conditions may have to be altered for different thermal cyclers.
The reverse transcription can be accomplished in 10-30 minutes at 61°C. The temperature range for annealing and extension will vary according to the properties of primers.
6. Collect the data and analyze results.
Troubleshooting
Refer to the table below to troubleshoot problems that you may encounter when you did RT-qPCR amplification with the kit.
Related Ordering Information
Caution
➤ During operation, always wear a lab coat, disposable gloves, and protective equipment.
➤ Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses.