Divya Prabhu

Jan 3, 2021

4 min read

Processing DNA and RNA from Whole Blood

DNA

Extracting DNA from Whole Blood

Step 1: Lysis

  1. First, mechanical disruption breaks open the cells. This can be done with a tissue homogenizer (like a small blender), with a mortar and pestle, or by cutting the tissue into small pieces.
  2. Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.

Step 2: Precipitation

  1. When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with mashed up cell parts.
  2. Precipitation separates DNA from this cellular debris.
  3. Next, alcohol (such as ethanol or isopropanol) is added and causes the DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.

Step 3: Purification

  1. Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to remove any remaining unwanted material and cellular debris.
  2. At this point, the purified DNA is usually re-dissolved in water for easy handling and storage.

RNA

Extracting RNA from Whole Blood

Step 1: Homogenization

  1. The pellet containing the supernatant is suspended in TRIzol. Then it is centrifuged at 12000 g for 10 minutes at 4 degrees.
  2. All proteins and nucleic acids in the supernatant are transferred to a new tube.

Step 2: Phase separation

Upper Aqueous Layer (RNA)
Interphase (DNA)
Bottom Organic Phase (proteins and lipids)
  1. Chloroform is added to the sample, and the sample is shaken vigorously and incubated for 3 minutes at room temperature.
  2. Now, the sample is centrifuged at 12000 g for 15 minutes.
  3. By the end of this step, you will see two layers- the upper colourless aqueous layer and the lower red organic layer (mostly phenol).
  4. The RNA in the aqueous layer is carefully removed and moved to a new tube.

Step 3: RNA Precipitation

  1. The upper aqueous layer is pipetted into a new tube, and isopropanol is added.
  2. It is centrifuged for 10 minutes at 12000 g at 4 degrees.

Step 4: RNA wash

  1. 75% of ethanol is poured onto the pellet and vortex it and centrifuge at 7500 g at 4 degrees for 5 minutes.
  2. The ethanol is removed via pipetting. The pellet is air-dried for 10 minutes and resuspended in DEPC-treated water.

References