I would like to perform a multiple displacement amplification experiment on microalgal cells（possibly diatoms, dinoflagellates, cryptophytes, gold algae, ciliates, etc.）.Due to transportation issues with the samples, I fixed the microalgal cells with glutaraldehyde. Could you please advise on an effective method for cell lysis of these fixed algae cells to adapt to the subsequent MDA reaction?
Glutaraldehyde is a commonly used fixative for preserving cell morphology and structure for downstream analysis, including whole-genome amplification (WGA) reactions. However, glutaraldehyde fixation can result in a decrease in DNA yield and quality, which could compromise the efficiency of WGA reactions. Here are some tips to help lyse cells fixed with glutaraldehyde and ensure compatibility with downstream WGA reactions:
1. Optimize the glutaraldehyde fixation protocol: The fixation process should be optimized for your specific sample type and downstream application. Fixation time, concentration, and temperature are all critical factors that can impact the quality and yield of DNA. The fixation time should be the minimum required to stabilize the cells. The most commonly used concentration is 0.1% to 2% glutaraldehyde in an isotonic buffer. The sample should be kept on ice during the fixation to reduce cross-linking of DNA.
2. Pre-treat fixed cells with enzymatic lysis buffers: Glutaraldehyde fixation can create crosslinks between proteins and nucleic acids, which can make it difficult to access the chromosomal DNA. Enzymatic lysis buffers, such as proteinase K and lysozyme, can help to break down these cross-links and improve lysis efficiency. Proteinase K, for example, cleaves peptide bonds, whereas lysozyme breaks down the cell wall of Gram-positive bacteria, allowing the lytic enzyme to access DNA. These enzymes should be used at high concentrations over long incubation periods.
3. Use mechanical lysis methods: High intensity ultrasonication, bead beating or grinding can help physically disrupt the cells, releasing nucleic acids from cross-linked proteins. However, care should be taken as these methods can damage DNA due to the increased shear forces on the DNA molecules.
4. Evaluate DNA quality before WGA: Before performing WGA on the lysed cells, it is essential to evaluate the quality and quantity of DNA. Different methods can be used to evaluate DNA quality, such as agarose gel electrophoresis, fluorescence-based assays like Qubit or Nanodrop.
5. Consider alternative fixation methods: Importantly, alternative fixative methods, such as formaldehyde, may be less likely to create cross-links in the DNA, allowing for more efficient WGA reactions.
Overall, optimizing the glutaraldehyde fixation protocol, using enzymatic and mechanical lysis methods, and evaluating DNA quality can help improve lysing efficiency for cells fixed with glutaraldehyde, thereby improving compatibility with downstream WGA reactions.
ultrasonication steps :
centrifugation of fixed cells for 30 min at 3500×g,
The pellet obtained is resuspended in 2mL of sterile water and homogenized. The cell suspension was then sonicated for 3 x 10 min on ice. The sample is left on ice for 1h, then centrifuged at 20,000×g for 30 min.
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