Processing DNA and RNA from Whole Blood
DNA stands for deoxyribonucleic acid. It is made up of units of biological building blocks called nucleotides.
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 DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins, so you are left with pure DNA.
The three necessary DNA extraction steps are:
Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside, and there are two ways to do this:
- 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.
- Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.
Step 2: Precipitation
- When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with mashed up cell parts.
- Precipitation separates DNA from this cellular debris.
- 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
- 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.
- At this point, the purified DNA is usually re-dissolved in water for easy handling and storage.
RNA, an abbreviation of ribonucleic acid, complex compound of high molecular weight that functions in cellular protein synthesis and replaces DNA (deoxyribonucleic acid) as a carrier of genetic codes in some viruses. Researchers widely use RNA to silence genes to learn something about their function.
Extracting RNA from Whole Blood
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulating, and expressing genes. Unlike DNA, RNA is more unstable due to the hydroxyl group's presence at the two prime position of the pentose ring.
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
- The pellet containing the supernatant is suspended in TRIzol. Then it is centrifuged at 12000 g for 10 minutes at 4 degrees.
- All proteins and nucleic acids in the supernatant are transferred to a new tube.
Step 2: Phase separation
- Chloroform is added to the sample, and the sample is shaken vigorously and incubated for 3 minutes at room temperature.
- Now, the sample is centrifuged at 12000 g for 15 minutes.
- By the end of this step, you will see two layers- the upper colourless aqueous layer and the lower red organic layer (mostly phenol).
- The RNA in the aqueous layer is carefully removed and moved to a new tube.
Step 3: RNA Precipitation
- The upper aqueous layer is pipetted into a new tube, and isopropanol is added.
- It is centrifuged for 10 minutes at 12000 g at 4 degrees.
Step 4: RNA wash
- 75% of ethanol is poured onto the pellet and vortex it and centrifuge at 7500 g at 4 degrees for 5 minutes.
- The ethanol is removed via pipetting. The pellet is air-dried for 10 minutes and resuspended in DEPC-treated water.
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