Hello all, I have been trying 5x Magmax 96 viral RNA isolation kit (AM1836-5) for extraction of total RNA and DNA samples from swab samples (assuming that the viral titer is too low). We have Magmax Express 96 magnetic particle procedure to automate the extraction procedure. So I prepare the plates as per user guide instructions and run the script using the instrument. Please note that I add a spike in RNA control in all of my samples and add two extraction controls in the plate with the spike in control and nuclease-free water instead of the sample to verify whether the extraction went okay. Following extraction, I run conventional PCR using primers for the spike in RNA control. I do quality check using NanoDrop and Qubit and the readings look okay. However, PCR did not work for any of the samples. No peak even for the spike in control in the tape station and Qiaxcel runs. Although PCR positive control worked perfectly and got the expected band size which likely indicates PCR worked okay. There might be something wrong in the extraction procedure which I can't figure out. I tried this process a couple of times with different concentrations of a spike in control (1 and 2 ul in lysis binding solution along with carrier RNA and buffer and isopropanol). Can you please enlighten me a bit about where might be the issue in the extraction process? Thanks a lot for you time and help!
Asma Sultana Based on the information you provided, it seems that you have followed the protocol and automation procedure correctly for the Magmax viral RNA isolation using the Magmax Express 96 system. However, you are experiencing issues with PCR amplification of the extracted RNA samples, specifically the spike in RNA control.
There could be several factors contributing to the PCR failure. Here are some potential areas to investigate:
1. Inhibition: It is possible that the extracted RNA contains inhibitors that are affecting the PCR amplification. These inhibitors can come from the sample matrix or be introduced during the extraction process. To address this, you can perform an inhibition control by adding known amounts of the extracted RNA to a PCR reaction that contains a known target. This will help you determine if inhibition is a problem.
2. RNA degradation: RNA is susceptible to degradation, especially in samples with low viral titers. Ensure that all reagents used during the extraction are RNase-free and that the samples are handled carefully to minimize degradation. Additionally, consider performing an RNA integrity assessment, such as using an Agilent Bioanalyzer or running a gel, to verify the quality and integrity of the extracted RNA.
3. Concentration of spike in control: The concentration of the spike in control may influence the efficiency of the extraction process. You mentioned trying different concentrations, but it may be worth optimizing this parameter further to ensure sufficient recovery of the spike in control RNA.
4. Sample quality and viral titer: It's important to consider the quality of the original swab samples and the expected viral titer. If the viral titer is indeed low, it may affect the success of the extraction and subsequent PCR amplification. Confirm the presence of viral RNA in your samples by using alternative detection methods like qPCR or digital PCR, if available.
5. PCR conditions: Double-check the PCR conditions, including primer design, annealing temperature, and cycling parameters. Ensure that the PCR protocol is appropriate for the target you are amplifying.
6. Troubleshooting with technical support: If you have tried the above suggestions and are still experiencing difficulties, consider reaching out to the technical support team of the Magmax viral RNA isolation kit or the instrument manufacturer. They may have specific troubleshooting steps or recommendations tailored to your situation.
Remember that troubleshooting experimental procedures can be a process of elimination, and it may require further optimization and testing to identify the root cause of the issue. Reviewing the protocol, confirming reagent integrity, and exploring potential sources of inhibition or degradation are important steps in diagnosing the problem.
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