Description
RT master mix for qPCR
GenieScript RT Master Mix for qPCR is a reverse-transcription master mix for fast preparation of qPCR-ready cDNA, with priming components included. It is built on a robust 4th-generation reverse transcriptase for fast, efficient cDNA synthesis, and includes a separate genomic DNA removal step.
The 4× GenieScript qRT Master Mix contains the reverse transcriptase, RNase inhibitor, dNTPs and a random/oligo(dT)20VN primer mix, so you only add template RNA and RNase-free water. Its high cDNA synthesis efficiency makes it a strong choice for low-input, low-expression or degraded RNA. The 5× gDNA Removal Mix completely removes genomic DNA contamination in 2 min, so intron-spanning primers are not needed. A No RT Control Mix is included for negative controls. The kit reverse-transcribes animal, plant and microbial RNA, and the cDNA is compatible with both dye-based and probe-based qPCR. For research use only.
Key Features
| High RT efficiency | Efficient cDNA synthesis gives lower Ct values in qPCR. | |
| Effective gDNA removal | Thoroughly removes 500 ng of genomic DNA to ensure accurate qPCR results. | |
| Broad template compatibility | Reverse-transcribes low-input, low-expression or degraded RNA efficiently. | |
| Fast workflow | Genomic DNA removal takes 2 min and reverse transcription 15 min. |
Specifications
| Format | 4× RT master mix with separate 5× gDNA Removal Mix |
| Primers | Random primer / oligo(dT)20VN primer mix (included) |
| RNA input | 1 pg - 1 μg total RNA per 20 μl reaction (up to 5 μg for low-expression targets) |
| Reaction time | gDNA removal 42°C 2 min; RT 37°C 15 min, 85°C 5 sec |
| RNA sources | Animal, plant and microbial RNA |
| Downstream | Dye-based and probe-based qPCR |
| Shelf life | 2 years |
Components
| Components | 100 rxns |
| RNase-free ddH2O | 2 × 1 ml |
| 5× gDNA Removal Mix | 300 μl |
| 4× GenieScript qRT Master Mixa | 500 μl |
| 4× No RT Control Mixb | 50 μl |
a. Contains GenieScript reverse transcriptase, RNase inhibitor, dNTP mix and random primer/oligo(dT)20VN primer mix. b. Identical to the 4× GenieScript qRT Master Mix but without reverse transcriptase; used to set up No RT control reactions.
Storage
Store at -30 ~ -15°C.
- Remove genomic DNA
- Mix the following in an RNase-free tube:
| Components | Volume |
| RNase-free ddH2O | To 15 μl |
| 5× gDNA Removal Mix | 3 μl |
| Template RNA | Total RNA: 1 pg - 1 μg |
- Pipette gently up and down to mix. Incubate at 42°C for 2 min.
- Prepare the reverse transcription reaction
- Add the master mix to the mixture from step 1 and pipette gently to mix:
| Components | Volume |
| 4× GenieScript qRT Master Mix | 5 μl |
| Mixture from step 1 | 15 μl |
No RT control (optional)
This negative control contains no reverse transcriptase and shows whether residual genomic DNA remains in the RNA template. Mix and pipette gently:
| Components | Volume |
| 4× No RT Control Mix | 5 μl |
| Mixture from step 1 | 15 μl |
- Run the reaction program
| Temperature | Time |
| 37°C* | 15 min |
| 85°C | 5 sec |
*For templates with complex secondary structure or high GC content, raise the temperature to 50°C to improve yield.
- Use or store the cDNA
- Use the cDNA for qPCR immediately or store at -20°C for 6 months. For long-term storage, aliquot and store at -70°C. Avoid repeated freeze-thaw cycles.
Q1: How much RNA can I use per reaction?
- Use 1 pg - 1 μg total RNA per 20 μl reaction. For low-expression targets, up to 5 μg can be used; more RNA can push Ct values outside the linear range.
Q2: Is the genomic DNA removal step required?
- No, it is optional. You can reverse-transcribe with the 4× GenieScript qRT Master Mix alone. When used, the 5× gDNA Removal Mix completely removes genomic DNA, so intron-spanning primers are not needed.
Q3: Can I use the cDNA for cloning or long-fragment PCR?
- No. The cDNA is intended for qPCR only and is not suitable for amplifying long fragments for cloning or other downstream applications.
Q4: How should I store the cDNA?
- Use it immediately or store at -20°C for 6 months. For long-term storage, aliquot and store at -70°C, and avoid repeated freeze-thaw cycles.
Q5: What is the No RT Control Mix for?
- It matches the master mix but lacks reverse transcriptase. A No RT control reaction shows whether residual genomic DNA remains in the RNA template.
