Extraction-free One-Step RT-qPCR Kit（Probe）

RT-qPCR is widely used in gene expression detection and quantification. Although traditional RT-qPCR has a one-step method, it involves sample RNA extraction, which is a cumbersome process with many influencing factors and very low detection throughput. This kit is an extraction-free one-step RT-qPCR detection kit. After cell washing, lysis buffer is directly added for lysis, and the lysate is directly used as a template for RT-qPCR amplification. The process from cell preparation to the end of PCR can be completed in less than two hours. The procedure is standardized, which greatly shortens the detection time, reduces the influencing factors, and significantly increases the detection throughput.

This detection kit uses the probe method (TaqMan probe). Before formal detection, each PCR parameter needs to be optimized and verified to ensure detection specificity and sensitivity. This detection kit does not include DNase treatment because DNase treatment may have incomplete digestion, and residual DNA templates may still affect later quantification. At the same time, DNase treatment may have incomplete inactivation, and residual DNase activity may affect the DNA quantification of later PCR amplification. Therefore, primer design needs to be based on RNA sequences, and the amplification products need to cover at least 2 exons to ensure that the amplification system cannot use DNA as a template for amplification, but can only use RNA as a template for amplification.

This kit consists of Cell Lysis Buffer L reagent, Amplification Buffer B reagent, and Amplification Enzyme mix E reagent. The specific specifications are as follows:

1. Cell preparation: The number of cells in the wells to be tested should be within the range of 25 to 15,000. For adherent cells, aspirate the culture medium, wash once with room-temperature DMEM serum-free medium, discard the washing solution, and then add cell lysis buffer. For suspension cells, centrifuge to aspirate the culture medium, wash once with room-temperature DMEM serum-free medium, discard the washing solution, and then add cell lysis buffer.

2. Cell lysis: Add 50 μl of reagent L to each well of a 96-well culture plate, and 10 μl of reagent L to each well of a 384-well culture plate. Tap the culture plate several times and shake at room temperature for 10 to 15 minutes. After using the cell lysate, it needs to be returned to 2–8°C for storage as soon as possible, and the low-temperature storage time should not exceed 8 hours. Long-term storage of cell lysates is not recommended.

3. RT-qPCR preparation: Prepare a 20 μl reaction system in the following order: 10 μl of reagent B, 4 μl of reagent E, 2 μl of primer-probe mix, 2 μl of cell lysate template, and 2 μl of enzyme-free water. It is not recommended that the cell lysate in the 20 μl reaction system exceed 2 μl.

4. RT-qPCR amplification: Reverse transcription at 42–50°C for 20–30 minutes, then 95°C for 1 minute. The qPCR parameters need to be determined based on the pre-experiment results of specific target genes and primer sequences. The following is an amplification setup for a housekeeping gene for reference:
Reverse transcription: 45°C for 20 minutes; then 95°C for 1 minute.
PCR amplification: 94°C for 20 seconds, 56°C for 20 seconds, 65°C for 20 seconds (collect fluorescent signals), 35–40 cycles; then 72°C for 3 minutes.

5. Perform melting curve analysis if necessary.

6. Result analysis.