Processing DNA and RNA from Whole Blood

DNA

DNA is a vitally important molecule for not only humans but most other organisms as well. DNA contains our hereditary material and our genes — it is what makes us unique.

Since DNA contains genetic code, much of it is studied and analyzed for a variety of reasons. Generally, DNA testing may be used to identify or classify family trees, and it may also be used in various sciences for study in diseases and crimes.

Extracting DNA from Whole Blood

The three necessary DNA extraction steps are:

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. Precipitation separates DNA from this cellular debris.
  2. 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. At this point, the purified DNA is usually re-dissolved in water for easy handling and storage.

RNA

Extracting RNA from Whole Blood

Several methods extract RNA from blood, but the most commonly used method is the Guanidium-thiocyanate-phenol-chloroform extraction method (TRIzol extraction). This method is a liquid-to-liquid extraction method.

Step 1: Homogenization

  1. 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. The ethanol is removed via pipetting. The pellet is air-dried for 10 minutes and resuspended in DEPC-treated water.

Just getting started. . .