It is important to ensure uniform distribution of lysis buffer throughout the sample. Proper mixing is especially important for viscous samples such as plasma, which can be difficult to handle and require more thorough mixing to ensure that the lysing buffer mixes uniformly. Insufficient mixing can lead to incomplete lysis of the viral particles, resulting in reduced DNA/RNA yields and compromised sensitivity of downstream assays.
During the binding step, mixing is necessary to ensure that the nucleic acids come in contact with the magnetic particles. To confirm proper mixing, visually inspect that the magnetic particles remain suspended as complete particle suspension is necessary for the wash buffer to be effective. Any remaining clumps will not be effectively washed (proteins are often the cause of particle clumping).
While the above principles apply to serum and plasma, you can learn more details in the "How to Automate Nucleic Acid Extraction" guide. In this comprehensive resource, you will find a video tutorial, guidance on experimental design for method development, and a troubleshooting guide that addresses each step.
Saliva samples may contain lower levels of viral RNA compared to other sample types, posing challenges for viral detection and downstream analyses. To maximize DNA/RNA yield, it may be required to optimize the extraction process, including adjustments to the lysis buffer and other components. In many cases, the observed reduction in yield compared to a functional manual control is attributed to inefficient binding, although poor elution may also be a contributing factor. Ensure that the correct volume of saliva sample is used during the extraction process, as using too little sample can result in diminished yield.
Saliva samples can be more variable than other sample types, with differences in viscosity, pH, and other factors that can affect the efficiency of nucleic acid extraction. When developing a method, try collecting a diverse set of samples to ensure your process can robustly handle the various range of samples.
Proteinase K digestions can help increase yield, improve lysis of capsid coated viral particles, and reduce protein burden in saliva, serum and other high-biomass biological samples. These viscous samples are protein-rich, leading to potential bead clumping issues. One solution is to include an additional wash step to reduce contamination and improve DNA/RNA purity. It is critical that the magnetic particles resuspend adequately during the wash steps. Adding additional wash steps can help if you encounter difficulties in particle resuspension.