The production of RNA isolation kits, RNA extraction methods and principles

RNA extraction is generally done using the Trizol method

Trizol is a total RNA extraction reagent that contains substances such as guanidine isothiocyanate, which can rapidly lyse cells and inhibit the nuclease activity released by cells. At present, the Trizol method is commonly used to extract RNA from tissues or cells.

How trizol works:

In homogenized or solubilized samples, Trizol® Reagent preserves RNA integrity while destroying cells and lysing cellular components. After chloroform centrifugation, the lysate is stratified into aqueous and organic phases. RNA is present in the aqueous phase.

After aqueous phase transfer, RNA is precipitated by isopropanol**. After removing the aqueous phase, DNA can be precipitated from the mesophase with ethanol, and protein can be obtained from the organic phase by adding isopropanol precipitation.

The current extraction kits all come with a matching instruction manual, and I can send them to you separately if needed.

The following are the materials that need to be prepared by Biodai centrifugation: absolute ethanol, normal saline, 1M NaOH, RNase-free centrifuge tubes.

1.Remove the precipitate and wash solution and proceed as follows:

a) Precipitate: add absolute ethanol; Add 9 ml to 51 ml of absolute ethanol.

b) Washing solution: 9ml add 21ml of absolute ethanol; Add 18 ml to 42 ml of absolute ethanol.

c) If there is a precipitate in the prepared precipitate, it can be dissolved in 37 and shaken well.

2.Sample Processing: a) Swab:

Add 800 L of normal saline to the RNase-free centrifuge tube, place the sample collected by the swab in normal saline, wash for 20 seconds to completely detach the cells, stick to the wall of the centrifuge tube and squeeze out the liquid on the swab, centrifuge at 12,000 rpm for 5 min, discard 700 L of supernatant, and the remaining 100 L of supernatant, and stir well with full shaking.

b) Sputum: Add 4 times the volume of 1 M NaOH to the collected sputum, leave at room temperature for 30 minutes, shake and mix every 10 minutes, centrifuge at 12,000 rpm for 5 min, discard the supernatant, add normal saline, mix well, centrifuge at 12,000 rpm for 5 min, discard 400 L of supernatant, and the remaining 100 L of supernatant, fully shake and mix.

3.Add 200 l of lysate and 20 l of digestion, mix with shaking and 56 water bath for 10 min.

4.Add 500 L of the precipitate, gently invert and mix well, if there is a translucent suspension, it will not affect the RNA extraction and subsequent experiments.

5.The adsorption column was put into the collection tube, the above solution was sucked into the adsorption column, let stand for 2 min, centrifuged at 12,000 rpm 4 for 1 min, and the waste liquid in the collection tube was discarded.

6.The adsorption column was placed in the ** header, 500 l of washing solution was added to the adsorption column, centrifuged at 12,000 rpm 4 for 1 min, and the waste liquid in the collection tube was discarded.

7.The adsorption column was placed in a ** header, centrifuged at 12,000 rpm 4 for 2 min, and the residual wash solution was removed.

8.Remove the adsorption column, place it in a new ml centrifuge tube, add 30-50 L of eluent, let it stand for 3 min, centrifuge at 12,000 rpm 4 for 2 min, and collect the RNA solution.

9.-70 saves, or directly for the next experiment.

Each kit has an instruction manual, and the instructions are as follows.

Dude: Don't know what you're using to extract?

Extracting different items is different.

Chloroform is an organic solvent with a relatively large molecular weight, and it can be used to effectively make the organic phase when extracting RNA.

and inorganic phases are quickly separated. DNA Extraction Process The organic phase is mainly composed of phenols and proteins, which detach the proteins and DNA and enter the aqueous phase. However, during RNA extraction, it is necessary to avoid the separation of protein and DNA, otherwise the DNA will be released into the aqueous phase.

Regardless of whether there is phenol or not, chloroform itself also has a denaturing effect on proteins, and it is easy to make the hydrophilic groups of DNA come into contact with water. So don't be drastic**.

The role of isopropanol is to protect the hydrophilic groups in the RNA or DNA strand through the hydrophobic action of -OH, which is equivalent to precipitation, but this reaction occurs in the aqueous phase and does not contradict the previous chloroform.

The role of chloroform has many aspects, one is to denature proteins as an organic solvent, precipitate them and remove them by centrifugation, and at the same time inhibit RNA activity through denaturation; Second, RNA extraction reagents usually contain phenol (the main ingredient of Trizol is phenol, many methods of preparing reagents also use phenol, and phenol will also be used when extracting proteins), phenol is slightly soluble in water, and there are traces of phenol in the aqueous phase after the extraction of alkane, if it is not removed, the nucleic acid will be damaged, and the phenol involved in the aqueous phase needs to be extracted through chloroform; Third, it is used as a solvent to extract some fat-soluble impurities (such as oils, fat-soluble pigments, etc.) in the sample, which plays a certain role in removing impurities.

Usually a small amount of isoamyl alcohol (1 25) will be added to the chloroform to reduce the foam produced by the protein due to ** during the denaturation process, which will affect the follow-up operation, but personal experience feels that there is no big difference between adding or not, maybe there is not much protein in my sample.

Isopropanol is used to precipitate nucleic acids and acts the same as ethanol. It's just that the dosage is a little less, which is enough, unlike ethanol precipitation, which requires at least 2V, and generally needs to double the volume. When there is a lot of aqueous phase, the volume of the centrifuge tube is limited, and too much ethanol can be added, isopropanol is generally used to precipitate.

However, I feel that the effect is not as good as ethanol, and occasionally there will be times when things cannot be precipitated.

Trizol is to lyse cells, chloroform is to extract RNA, isopropanol precipitates RNA, and finally cleaned with alcohol, impurities may be introduced during the extraction of the upper layer, or they may be introduced during the operation.

The main substance of Trizol is guanidine isothiocyanate, which destroys cells so that RNA is released while protecting the integrity of RNA . After chloroform was added and centrifuged, the sample was divided into an aqueous layer and an organic layer. RNA is present in the aqueous layer.

After collecting the upper layer of aqueous samples, the RNA can be reduced by isopropanol precipitation. After the aqueous layer is removed, the DNA and protein in the sample can also be reduced by precipitation. Ethanol precipitation can precipitate DNA from the middle layer, and the addition of isopropanol to the organic layer can precipitate proteins.

The RNA I just mentioned, this is in the program I wrote.

1. RNase-free sterilized water DPC water: use distilled water in a glass bottle (180 2h) baked at high temperature, and then add DEPC (volume volume), treat overnight and then autoclave.

DEPC Water:

Preparation of DEPC water for soaking the experimental utensils: add 1ml of DEPC to 1000ml of double distilled water, put it in a 1000ml volumetric flask and let it stand for 4 hours before setting aside.

Preparation of DEPC water with 75 ethanol: 100ml saline bottle filled with 40ml double distilled water, add 40ul DEPC, 37 overnight, and send to high pressure.

Ethanol: Prepare 75% ethanol with DEPC-treated water (prepared in autoclavable vessels) and then store in a low-temperature freezer in a glass bottle baked at high temperature.

Preparation of 75% ethanol.

One part sterile, enzyme-free water is mixed with 3 parts (volume) of absolute ethanol. 3. Absolute ethanol.

4. Chloroform (preferably new).

5. Isoamyl alcohol (preferably new).

I usually buy trizol reagent, and I really haven't matched it.

In addition to Trizol, chloroform, isopropanol, ethanol, depc-treated water are required