TRIzol reagent

TRIzol reagent is the type of reagent which is widely used by scientists for the complete precipitation of high-quality RNA from cell or tissue samples. Also, it is used to precipitate the volume of DNA and protein from the cells or tissue with great efficiency. It is a monophasic solution that consists of the guanidinium isothiocyanate and phenol within one phase (also called a true homogenous solution) that is used for the denaturation of proteins and the precipitation of the biological materials from the sample. Studies suggest that it is the recently developed method that is generally used for the solubilization and extraction of DNA, RNA, and proteins from cells. Especially this method is more reliable when there is a huge amount of RNase present within the cell or when it is difficult to separate the nuclear RNA from the cytoplasmic RNA in the cell. It is because the TRIzol reagent can inhibit the activity of RNase enzymes during the dissolution of cellular components. When the number of cells is homogenized with the TRIzol reagent. Then upon addition of the chloroform, these biological materials undergo separation into different phases of the solution. RNA begins to precipitate in the aqueous phase, DNA begins to precipitate into the Inter or organic phase, while protein precipitates within the organic layer of the solution. That’s why it is called TRIzol reagent because it can be used to precipitate these biological materials from a single sample within a very short period. It is also an effective method for the precipitation of the very small amount of RNA within the sample, for example, endogenous RNAs, micro RNA, and piwi-associated RNAs. Somehow this procedure is expensive and difficult to perform because of the difficult resuspension of the RNA pallet.

An RNA extraction method using the TRIzol reagent gaining popularity nowadays because of its reliability along with the short time which is required for purification than the conventional method. This development is necessary because of the advancement in clinical research that’s required high-quality RNA. As we have already discussed that the RNA which is isolated using TRIzol reagent remains in the aqueous phase while the DNA or proteins are in the organic phase or DNA in the interphase. Scientists use several solutions to precipitate these biological entities from these phases. For example, isopropanol use used for this purpose to precipitate RNA from the aqueous phase. Similarly, ethanol can be used for the precipitation of the DNA from the interphase and isopropanol for the precipitation of the proteins from the organic layer. It is very helpful for the isolation of these biological materials from small quantities of tissues i.e. 50-100mg and large samples of the tissue (≥1g). All these procedures require only 1 hour because of their greatest efficiency. After isolation of RNA, it may be used in several techniques for further studies, that includes PCR, RNase protection-assay, northern blot, etc.

On the other hand, several tissues like the rat pancreas have enriched amounts of nucleases, which in return produce difficulty in the isolation of the intact RNA from the samples. Similarly, calf thymus tissues cause trouble during RNA isolation because, during the aqueous phase transfer, there will be a continuous carryover of the interphase. This plague can be prevented by using the TRIzol reagent. This will cause the precipitation of the RNA 6-times more than the single-step RNA isolation method.

Figure 1:Representation of gel analysis of the isolated RNA by TRIzol reagent. Line-1: pancreas of rat; Line-2: calf thymus

Similarly, as we discussed earlier that rat pancreas tissue has a huge number of nucleases enzymes, that affect the isolation of RNA using the single-step RNA isolation method. Studies recommend the use of TRIzol reagent for this purpose. They suggested that the TRIzol reagent has a resistant activity for the nucleases enzymes that will allow the lysis of cellular components without interfering with the RNA.  First, the rat pancreas tissue is grounded, then collected within 1 ml of liquid Nitrogen. After that, this sample is then transferred into the 1ml of TRIzol reagent for further processes. After that this extracted RNA is treated with DNase 1 and then to evaluate the extracted RNA, we checked the specific genes for the pancreas including, amylase, insulin 1, glucagon, etc along with the expression of beta-actin by using the RT-PCR.

Figure 2: Analysis by RT-PCR of isolated RNA from conventional and grinding group of tissue sample, to check the profile of RNA by checking cDNA of insulin 1 and glucagon.

As we have discussed above that the TRIzol reagent is used to isolate RNA, proteins, and DNA from different samples of cells or tissues.  As these amounts of extracted DNA, RNA, and proteins are further used for the transcription and expression analysis of the proteins. Many of the studies reported TRIzol reagent used for the proteins and RNA extraction. However, few studies reported that the TRIzol reagent can be used for the extraction or isolation of DNA. This is because of its time-consuming demerit for DNA extraction along with the impurity regarding its extraction. Scientists overcome this trouble by extracting the DNA with TRIzol reagent with the help of a silica column. It has more advantages than any other technique. One of the major advantages is that this reagent is very stable, and can be maintained at a very low temperature without making changes in the profile of nucleic acids either DNA or RNA. Similarly, this reaction can be performed at room temperature, so there is no need for a warm bath. Hence, it also reduced the cost of the experiment. Moreover, it is protease free, and inexpensive, because it does not require the use of magnetic bead matrices, incubator, and proteinase K enzyme. Also, it gives results in a very short period.

So, despite the use of TRIzol reagent to extract DNA and RNA, it can be used for the extraction of proteins. This reagent can extract intact proteins from the samples. During the extraction, a major problem is the stability of the proteins, which can be controlled by processing them at a very low temperature with a short processing period or by using protease inhibitors. During the extraction of proteins by using TRIzol reagent, it disabled the protease activity because of its constituents which include phenol and guanidine isothiocyanate.