Extraction-free One-Step RT-qPCR Kit（SYBR Green）

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 dye method (SYBR Green). 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 incomplete digestion may occur in DNase treatment, and residual DNA templates may still affect subsequent quantification. At the same time, incomplete inactivation may occur in DNase treatment, and residual DNase activity may affect the DNA quantification in subsequent 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 in the range of 100 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 use, the cell lysate needs to be stored at 2–8°C 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 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 at 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, 62°C for 20 seconds, 70°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.

1. Primer Design: It needs to be based on the RNA sequence, and the amplification product must cover at least 2 exons to ensure that the amplification system cannot use genomic DNA as a template for amplification, but only RNA. This can be achieved under the following circumstances: a. The intron between the two exons is relatively long, so the length of amplification using the genome as a template significantly exceeds the length that the polymerase in the system can complete within the amplification extension time; b. The intron between the two exons is relatively short, but at least the 2 to 5 bases at the 3' end of one primer and the remaining bases on the side of its 5' end are distributed on different exons and are different from the base sequence at the 5' end of the adjacent intron (refer to the figure below), making amplification using DNA as a template impossible. If necessary, please contact product technical support for assistance.

2. PCR Amplification Parameters and System: For any PCR and any pair of amplification primers, pre-experiments are required to determine the optimal amplification temperature and time settings, primer amount, enzyme amount, and even Mg²⁺ concentration adjustment, which are crucial for detection specificity and sensitivity. It is necessary to conduct pre-experiments similar to cell number gradient detection, and start the microplate high-throughput experiment only after obtaining a good linear relationship.

3. Melting Curve: In the dye-based amplification system, any specific or non-specific amplification products, including primer dimers, can give amplification signals. Chemical substances from different cells or cell treatments may also affect PCR amplification. Melting curve analysis can perfectly distinguish between specific and non-specific amplification as well as primer dimers. Melting curve analysis needs to be used in the process of determining experimental parameters in pre-experiments.