Flow Cytometry Technical Resources

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In this article, the following topics will be covered:

The genomic DNA (gDNA) and total RNA are frequently the genetic elements targeted for molecular biology experiments, since these are the main sources of genetic information in an organism.

Genomic DNA and total RNA extraction have a straightforward methodology, requiring only cell lysis to accomplish the task. However, it is recommended to consider the type of organisms you are using in your lab research. Some organism’s cells have a cell wall structure, which confers extra protection to cells. This is the case for most yeast, plants and bacteria species. For these organisms, it is recommended to include an extra step for cell lysis before disrupting the plasma membrane. This step is usually an enzymatic or mechanical process to disrupt the cell wall. The purification of DNA and RNA molecules is required to avoid contaminations with other intracellular components, such as proteins and metabolites. Some commonly used methods for DNA and RNA purification are precipitation with phenol-chloroform or isopropanol, or by spin columns with silica membrane.

However, if your target DNA is a plasmid, you should adapt your procedure to make sure the plasmid DNA (pDNA) is separated from gDNA. Alkaline lysis is the most current protocol used in the labs. The lysis buffer has in its composition sodium hydroxide and SDS, which will be responsible for denaturation of gDNA and pDNA. Subsequently, a neutralization buffer is added to renature the pDNA but not the gDNA. Because of the size of gDNA, it is much more difficult to renature again, unlike the pDNA. Then the same procedure of gDNA purification can be used for pDNA purification.

If your research is focused on gene expression analysis, then you should target total RNA. The same procedure used to extract and purify gDNA can be used for total RNA purification, although some additional steps are required, as you will see in the following RNA extraction and purification protocol. Boster recommends the following protocols for sample preparation depending on your initial sample source.

DNA Extraction and Purification

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Reagents:

  • lysis buffer: 1% SDS, 0.5 M NaCl
  • isopropanol
  • 70% (v/v) ethanol
  1. Collect the cells for DNA extraction into 2 ml tubes.
  • Note: If you are using cell suspensions you can move directly to the lysis procedure. If you are using samples, like cell tissues that are difficult to homogenize, you can use liquid nitrogen to create a homogeneous cell suspension. At the time you have a cell suspension, you should be careful about the membrane structure of your sample. Usually, for cells with cell wall, it is recommended a pre-lysis treatment to degrade this barrier. You can use an enzymatic method or a mechanical method.
  1. Add 1ml of cell suspension into 2 ml tubes
  2. Add 100 mg of 0.5 mm glass beads and vortex during 15 min.
  • Note: Alternatively, you can use 10U of lyticase (for fungi samples) or 10U of lysozyme (for gram-negative bacteria).
  1. Spin the cell suspension at top speed for 1 min using a centrifuge,
  2. Discard the supernatant and resuspend in 1000 μl of the lysis buffer.
  3. Spin at top speed for 1 min and  
  4. Transfer the supernatant into a new 2ml tube.
  5. Add 500 μl of isopropanol and mix gently.
  6. Place the mixture on ice for 5 min.
  7. Spin at top speed 1min,
  8. Discard the supernatant and wash the DNA pellet with 500 μl 70% (v/v) ethanol.
  9. Spin again at top speed during 1 min.  
  10. Discard the supernatant.
  11. Let the pellet air-dry, placing the 2 ml tubes upside down in a paper-towel.
  12. Dissolve the DNA in 50 μl ultrapure DEPC H2
  13. Use the Nanodrop equipment to access DNA concentration and quality.
  • Note: Nucleic acids absorb UV light at 260 nm, whereas proteins and phenolic compounds absorb at 280 nm. Many organic compounds as well as phenol, TRIzol, and chaotropic salts, have strong absorbances at around 230 nm.   The A260/280 ratio is used to identify protein contamination. For RNA and DNA, A260/280 ratios should be around 2.1 and 1.8, respectively. The A260/230 ratio indicates the presence of organic contaminants, such as phenol, TRIzol, chaotropic salts and other aromatic compounds. Samples with 260/230 ratios below 1.8 have a significant amount of these contaminants.
  1. Store the DNA sample at 4 °C for immediate use or −20 °C for long-term storage.

RNA Extraction and Purification

*Note: During this protocol always use new gloves and RNase free material.

  1. Collect the cells into 2 ml tubes.
  • Note: In the case you are using cell suspensions you can move directly to the centrifuge step before addition of lysis buffer. Although if you are using samples, like cell tissues that are difficult to homogenize, you can use liquid nitrogen to create a homogeneous cell suspension. For samples, in which cells have cell wall, add 1 ml of cell suspension into 2 ml tubes and add 100 mg of 0.5 mm glass beads and vortex for 15 min.
  1. Spin the cell suspension at top speed for 1 min using a centrifuge,
  2. Discard the supernatant and resuspend in 1 ml of lysis buffer prewarmed at 65 ºC.
  3. Add 900 μl of acid phenol: chloroform and vortex for 10 s.
  4. Let the 2 ml tube in the bench for 10 min at room temperature.
  5. Spin at top speed for 10 min at 4 °C
  6. Transfer the supernatant to new 2 ml tube
  7. Add 0.3 volume of 5 M sodium acetate and 0.7 volume of acid phenol:chloroform.
  8. Mix gently the tube and incubate on ice for 10 min.
  9. Top spin for 10 min at 4 °C
  10. Transfer the supernatant to fresh 2 ml tubes.
  11. Add 0.1 volume of 3 M sodium acetate and the same volume of isopropanol.
  12. Incubate the tubes at -20 °C for 1 h.
  13. Spin at top speed for 10 min at 4 °C and discard the supernatant.
  14. Wash the pellet with 500 μl ethanol (70 %)
  15. Centrifuge at 7,500 g for 5 min at 4 °C.10.
  16. Air dry the pellet for 5–10 min
  17. Dissolve in 25 μl RNase free water.
  • Note: For further use of the RNA for expression analysis, it is highly recommended to treat the RNA sample with DNase, an enzyme that digests DNA.
  1. Add into a 1ml tube 10ug of RNA,
  2. Add 5 μl of DNase buffer,
  3. Add 1 μl of DNase and complete the volume up to 50 μl with RNase free water.
  4. Incubate at 37 ºC for 30 min.
  5. Add .500 μl ethanol (70 %)
  6. Centrifuge at 7,500 g for 5 min at 4 °C.10.
  7. Air dry the pellet for 5–10 min
  8. Use the nanodrop equipment to access RNA concentration and quality
  9. Store the RNA sample in 25 μl RNase free water at 4 °C for immediate use or −20 °C for long-term storage.
  • Note: In order to remove DNase, that can destroy cDNA molecules in further qRT-PCR experiments, add 1ul of DNase inhibitor.