The CD One Step qRT-PCR Probe Kit is specifically designed for qPCRs that use RNA as a framework (such as RNA virus). Reverse transcription and PCR can both be completed in one tube, lowering the amount of pipetting steps and the risk of contamination. The 2 One Step Q Probe Mix includes an optimized buffer and dNTP/dUTP mix, making it ideal for high-sensitivity detection systems using fluorescence-labeled probes (such as TaqMan®). One Step Q Probe Enzyme Mix contains reverse transcriptase and hot-start Taq DNA Polymerase, allowing for highly sensitive total RNA detection (as low as 0.1 pg of total RNA or RNA with less than 10 copies). A small amount of RNA can be detected and quantified using the two enzymes in combination with an optimized buffer system. Since it is tolerant of target sequence diversity, this kit can discern various types of RNA with various sequences.
Storage:
All components should be stored at -20℃.
Components:
Specifications:
| Sample amount | total RNA as little as 0.1 pg or RNA with less than 10 copies |
| Sample type | RNA |
1. Prepare the reaction solution in a RNase-free PCR tube as follows:
| RNase free ddH2O | to 20 μl |
| 2x One Step Q Probe Mix | 10µl |
| One Step Q Probe Enzyme Mix | 1µl |
| 50x ROX Reference Dye 1 | 0.4µl |
| Gene Specific Primer Forward (10 μM) a | 0.4µl |
| Gene Specific Primer Reverse (10 μM) | 0.4µl |
| TaqMan Probe (10 uM) b | 0.2µl |
| Template RNAc | Total RNA: 1 pg-1 μg |
Note: For each component, the volume can be adjusted according to the following principle:
a. The final concentration of primer is usually 0.2 μM, and if necessary, it can be adjusted between 0.1 μM and 1.0 μM.
b. The final concentration of TaqMan Probeprimer can be adjusted between 50 nM and 250 nM.
c. The accuracy of template volumes significant impacts on the qPCR results, due to the high sensitivity this kit. Therefore, to improve experimental repeatability, it is recommended to dilute the template and pipet more volume to the reaction system.
2. Place the sample in a qPCR instrument and run the following program for One Step qRT-PCR:
Standard Program
| Stage 1 | Reverse Transcription | Reps: 1 | 50°C a | 15 min |
| Stage 2 | Pre-denaturation | Reps: 1 | 95°C | 30 sec |
| Stage 3 | PCR Cycles | Reps: 45 | 95°C | 10 sec |
| 60°C | 30 sec b |
Fast Program (suitable for most One Step qRT-PCR)
| Stage 1 | Reverse Transcription | Reps: 1 | 50°C | 5 min |
| Stage 2 | Pre-denaturation | Reps: 1 | 95°C | 30 sec |
| Stage 3 | PCR Cycles | Reps: 45 | 95°C | 5 sec |
| 60°C | 20 sec c |
Note:
a. For templates with complex secondary structure or high GC-content, the temperature can be increased to 55℃, which will improve the sensitivity and performance.
b. The extension time varies between different qPCR instruments used. For ABI 7700 and 7900HT, the extension time should be ≥ 30 sec; for ABI 7000 and 7300, the extension time should be ≥ 31 sec; for ABI 7500, ≥ 34 sec; and for ABI StepOne Plus, ≥ 10 sec.
c. Please check the fast program is compatible for the qPCR instrument.
Tips:
- The 2× One Step Q Probe Enzyme Mix contains glycerol. Before pipetting, please collect the liquid by a brief centrifugation.
- To avoid RNase contamination, please keep the experiment area clean, wear clean gloves and masks, and use RNase-free tubes and tips.
