RNA isolation is a crucial step in gene expression analysis and molecular biology research. The isolation process involves separating RNA molecules from cellular components to obtain pure RNA samples for downstream applications. TRIzol protocol, also known as the "guanidine thiocyanate-phenol-chloroform protocol," is a standard method used for RNA isolation. This protocol works by disrupting cells and denaturing RNases, allowing RNA to be extracted from the cell. In this blog, we will discuss the TRIzol RNA isolation protocol in detail.

TRIzol RNA Isolation Protocol

The TRIzol protocol has been widely used for RNA isolation from various biological samples, including tissues, cells, and microorganisms. The protocol involves the following steps:

1. Collecting samples: Collect the biological sample, such as tissue or cells, in a sterile container. Keep the sample on dry ice or in liquid nitrogen while transporting it to the lab.

2. Homogenization: Homogenize the biological sample to break down the tissue and release the cells. The sample can be homogenized using a homogenizer or a mortar and pestle. Be careful not to over homogenize the sample as this can result in RNA fragmentation.

3. Adding TRIzol reagent: Add TRIzol reagent to the homogenized sample at a ratio of 1 ml of TRIzol per 50-100 mg of tissue or 10^7-10^8 cells. Mix the sample by vortexing for 15-30 seconds to ensure complete mixing of the sample with the reagent.

4. Phase separation: Incubate the sample at room temperature for 5 minutes to allow the TRIzol reagent to dissolve the cellular membranes and denature RNases. Centrifuge the sample at 12,000 g for 15 minutes at 4°C to separate the sample into three phases: a lower organic phase, an interphase, and an upper aqueous phase.

5. RNA extraction: Carefully remove the upper aqueous phase containing RNA molecules and transfer it to a new tube. Add an equal volume of isopropanol to precipitate the RNA molecules. Mix the tubes by inverting them several times and incubate it for 10 minutes at room temperature.

6. RNA washing: Centrifuge the tubes at 12,000 g for 10 minutes at 4°C to pellet the RNA. Wash the RNA pellet with 75% ethanol to remove any remaining salts and contaminants. Centrifuge the tubes again and discard the supernatant.

7. RNA resuspension: Air dry the RNA pellet and resuspend it in RNase-free water or buffer.

Tips for TRIzol RNA isolation

- Always use RNase-free techniques and reagents to avoid RNA degradation.
- Choose the appropriate TRIzol-to-sample ratio depending on the cell or tissue type and the amount of RNA required.
- Avoid over homogenization of the sample, which can lead to RNA fragmentation.
- Remove any leftover tissue or cellular debris after phase separation to obtain a pure RNA sample.
- Avoid vortexing the RNA pellet after washing or resuspension, as it can cause RNA shearing.

Conclusion

TRIzol RNA isolation protocol is a reliable and widely used method for RNA extraction from various biological samples. The protocol involves the disruption of cells and the denaturation of RNases, allowing the extraction of pure RNA from the sample. This protocol, when followed correctly, can provide high-quality RNA samples for downstream applications like RT-PCR, RNA sequencing, and gene expression analysis.

In summary, TRIzol RNA isolation protocol is an ideal protocol for RNA isolation from cells and tissues. If you are performing gene expression analysis or other molecular biology experiments that rely on pure RNA, then this protocol is a must-try. Follow the protocol carefully to ensure a good yield, quality, and purity of RNA samples for downstream applications.