SYBR Green I-based qPCRs that instantly use RNA (such as RNA virus) as templates are designed with the CD One Step qRT-PCR SYBR Green Kit. Reverse transcription and PCR can both be completed in one tube, lowering the amount of pipetting steps and the risk of contamination. The kit employs proprietary technology to deliver accurate, sensitive, and repeatable monoplex or multiplex qRT-PCR, as well as a large quantitative area for a perfect standard curve and compatibility with a variety of real-time instruments.
Storage:
All components should be stored at -20℃ and protected from light.
Components:
Specifications:
| Sample amount | total RNA as little as 1 pg |
| Sample type | RNA |
1. Prepare the reaction solution in a RNase-free centrifuge 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 |
| Template RNA b | 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 uM and 1.0 μM.
b. The accuracy of template volumes significant impacts on the qPCR results, due to the high sensitivity of 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:
| Stage 1 | Reverse Transcription | Reps: 1 | 50°C a | 3 min b |
| Stage 2 | Pre-denaturation | Reps: 1 | 95°C | 5 min |
| Stage 3 | PCR Cycles | Reps: 40 | 95°C | 10 sec |
| 60°C | 30 sec c | |||
| Stage 4 | Melting Curve | Reps: 1 | Default |
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 time for reverse transcription can be extended to 15 min, which will benefit the yield of cDNA.
c. 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.
Tips:
- The One Step SYBR Green Enzyme Mix contains glycerol. Therefore, before pipetting, please collect the liquid by a brief centrifugation.
- Vortex the 2× One Step SYBR Green Mix before pipetting, and protect it from light.
- To avoid RNase contamination, please keep the experiment area clean, wear clean gloves and masks, and use RNase-free tubes and tips.
