TW201425325A - A method for purifying microRNA and totalRNA molecules - Google Patents

Abstract

The present invention relates to methods, kits, and compositions for purifying microRNA and totalRNA molecules. In particular, the present invention provides methods for purifying microRNA and total molecules from a sample containing both microRNA molecules and larger RNA molecules using Acid Phenol and Guanidine Thiocyanate, and a RNA binding matrix comprising glass fiber and plastic column, as well as compositions and kits for practicing such methods. In certain embodiments, the samples comprise animal and plant, further comprise monolayer cells, suspension cells, biological fluids (whole blood, plasma, serum, buffy coat, CSF, semen, saliva, biological fluids containing RNA virus, and body fluids), and tissues.

Description

MicroRNA and total RNA extraction method

The present invention relates to the fields of inorganic chemistry, organic chemistry, molecular biology, cell biology, biochemistry, and medicine. More particularly, the present application discloses a method for extracting microRNAs and total ribonucleic acids from a single layer of animal or plant cells, suspended or plant cells, biological fluids or body fluids, animal or plant tissues.

RNA is a powerful biological material. It is an active molecule that is directly involved in the execution or control of multiple biological functions. Different types of ribonucleic acids exist in various cellular processes of living organisms, and are the key to human development of new diagnostic methods and research and treatment of diseases.

A microRNA microRNA is a non-coding ribonucleic acid, that is, a ribonucleic acid that does not translate a protein. In vivo, non-coding ribonucleic acid plays a very important role in controlling gene expression, signaling, and regulating protein activity. MicroRNAs were first discovered in the nematode in 1993. They are a group of short-stranded ribonucleic acids that fold themselves and are constructed like hairpins. According to bioinformatics, at least one-third of the genes in the human body are regulated by micro-ribonucleic acid. More than 600 micro-ribonucleic acids have been found in mammals and humans, and it is possible to judge the large-scale use of micro-ribonucleic acids in organisms for gene regulation. . Therefore, the detection of microRNA performance in humans is an increasingly important research work in the future.

The detection of microribonucleic acid requires the extraction of small RNA fragments in the cell. Currently, the ribonucleic acid extraction kits on the market are generally divided into two major types of extraction kits, total RNA and miniribonucleic acid. It is also possible to extract the extraction set of microribonucleic acid at the same time. At the same time, most users still use phenol and chloroform for microRNA extraction. Therefore, the development of a multifunctional and safe glyco-nucleic acid extraction kit is the field. The relevant manufacturers are eager to study the direction of development.

Based on the above objectives, the present inventors collected relevant materials and, through continuous testing and modification, developed an extraction method capable of simultaneously extracting total ribonucleic acid and microribonucleic acid.

The main object of the present invention is to rapidly destroy cells by using acidic phenol, and at the same time, effectively inhibit the RNase released by the cells during the extraction process, and avoid degradation of the ribonucleic acid. At the same time, the use of guanidinium isothiocyanate causes adsorption of ribonucleic acid with glass fibers, etc., whereby the ribonucleic acid can be rapidly extracted using a centrifugal operation tube.

The secondary object of the present invention is to utilize acidic phenol (pH 4~6), and selectively retain only ribonucleic acid (RNA) during the extraction process, thereby eliminating deoxyribonucleic acid (DNA) and avoiding the extraction product. Oxygen ribonucleic acid contamination.

The invention does not need to add chloroform in the whole process, and can greatly improve the operation safety of the user.

The present invention provides a decomposition solution and a centrifugal operation tube for simultaneously extracting microribonucleic acid and total ribonucleic acid, and a method for simultaneously extracting microribonucleic acid and total ribonucleic acid.

It must be noted that the singular "a" or "an"

The invention will now be described in detail with reference to the following specific non-limiting examples.

Example 1: Extraction of microRNAs and total RNA from osteosarcoma cells

First, break the osteosarcoma cells (the appropriate number of cells is 1x10 ⁴ -5x10 ⁶ ), and add 100 μl/cm ^{2 to the} cell plate using a decomposition solution (VRX buffer, containing acid phenol, guanidinium isothiocyanate) (Refer to the table) 1) The VRX buffer is thoroughly mixed with the cells, transferred to a centrifuge tube containing no RNase, and the shock completely ruptures the cells. The pellet was removed by centrifugation at 10,000 xg for 2 minutes and the supernatant was transferred to a new RNase-free centrifuge tube.

From Table 1, it is known that 400 μl of VRX buffer is added to the cells in each well of a 12-well plate.

98-100% added one volume of isopropanol to the sample, vortexed well, transferred to miTotal ^TM RNA Column and centrifuged for one minute, centrifuged down the liquid discarded, and added 500 μl WS Buffer, centrifuged one minute After the liquid was poured off, the action was repeated and the WS buffer was added twice in total. After centrifugation for three minutes, spun down to ensure that the liquid completely, the miTotal ^TM RNA Column moved to the new RNA enzyme-free centrifuge tube. Add 50μl of the enzyme RNA-free water to the miTotal ^TM RNA Column, centrifuged at full speed for one minute, i.e., to obtain a micro RNA (of microRNA) and total RNA (totalRNA). Please save this sample at minus 70 degrees immediately. The result is shown in Figure 1.

Example 2: MicroRNA and total RNA extracted from human whole blood

Prepare 100 μl of biological fluid (white blood cells, whole blood, plasma, serum, yellow clot, cerebrospinal fluid, semen, saliva and body fluids)

First add the sample to 100 μl (1 volume) PBS and 300 μl (three volumes) of VRX buffer, mix thoroughly and shake, centrifuge 10,000 xg for 2 minutes to remove the precipitate, and transfer the supernatant to a new one. In an RNase-free centrifuge tube. If the supernatant is whole blood or plasma, be careful of the red blood cell layer during the transfer.

98-100% added one volume of isopropanol to the sample, vortexed well, transferred to miTotal ^TM RNA Column and centrifuged for one minute, centrifuged down the liquid discarded, and added 500 μl WS Buffer, centrifuged one minute After the liquid was poured off, the action was repeated and the WS buffer was added twice in total. After centrifugation for three minutes, spun down to ensure that the liquid completely, the miTotal ^TM RNA Column moved to the new RNA enzyme-free centrifuge tube. Add 50μl of the enzyme RNA-free water to the miTotal ^TM RNA Column, centrifuged at full speed for one minute, i.e., to obtain a micro RNA (of microRNA) and total RNA (totalRNA). Please save this sample at minus 70 degrees immediately. The result is shown in Figure 2.

Example 3: Extraction of microRNAs and total RNA from spleen and liver tissues

Prepare 50 mg of tissue separately, add 500 μl of VRX buffer, homogenize the sample completely using a homogenizer, remove the pellet by centrifugation at 10,000 xg for 2 minutes, and transfer the supernatant to a new RNase-free centrifuge tube. In this case, the purification steps of microRNAs and total RNAs can be continued.

If the sample is already present in VRX buffer, the ratio is approximately 1:10 (sample: VRX) and the volume of VRX buffer should not be less than 10%.

98-100% added one volume of isopropanol to the sample, vortexed well, transferred to miTotal ^TM RNA Column and centrifuged for one minute, centrifuged down the liquid discarded, and added 500 μl WS Buffer, centrifuged one minute After the liquid was poured off, the action was repeated and the WS buffer was added twice in total. After centrifugation for three minutes, spun down to ensure that the liquid completely, the miTotal ^TM RNA Column moved to the new RNA enzyme-free centrifuge tube. Add 50μl of the enzyme RNA-free water to the miTotal ^TM RNA Column, centrifuged at full speed for one minute, i.e., to obtain a micro RNA (of microRNA) and total RNA (totalRNA). Please save this sample at minus 70 degrees immediately. The result is shown in Figure 3.

Example 4: Extracting micro-RNAs and total RNA from golden dew

A 50 mg plant sample was prepared and ground to a fine powder using liquid nitrogen and then transferred to a new centrifuge tube. 500 μl of VRX buffer was added and the sample was completely homogenized using a homogenizer. The pellet was removed by centrifugation at 10,000 xg for 2 minutes and the supernatant was transferred to a new RNase-free centrifuge tube.

If the sample is already present in VRX buffer, the ratio is approximately 1:10 (sample: VRX) and the volume of VRX buffer should not be less than 10%.

98-100% added one volume of isopropanol to the sample, vortexed well, transferred to miTotal ^TM RNA Column and centrifuged for one minute, centrifuged down the liquid discarded, and added 500 μl WS Buffer, centrifuged one minute After the liquid was poured off, the action was repeated and the WS buffer was added twice in total. After centrifugation for three minutes, spun down to ensure that the liquid completely, the miTotal ^TM RNA Column moved to the new RNA enzyme-free centrifuge tube. Add 50μl of the enzyme RNA-free water to the miTotal ^TM RNA Column, centrifuged at full speed for one minute, i.e., to obtain a micro RNA (of microRNA) and total RNA (totalRNA). Please save this sample at minus 70 degrees immediately. The result is shown in Figure 4.

Example 5: Extraction of micronuclei (microRNA) and total ribonucleic acid (total RNA) from tobacco

A 50 mg plant sample was prepared and ground to a fine powder using liquid nitrogen and then transferred to a new centrifuge tube. 500 μl of VRX buffer was added and the sample was completely homogenized using a homogenizer. The pellet was removed by centrifugation at 10,000 xg for 2 minutes and the supernatant was transferred to a new RNase-free centrifuge tube.

If the sample is already present in VRX buffer, the ratio is approximately 1:10 (sample: VRX) and the volume of VRX buffer should not be less than 10%.

98-100% added one volume of isopropanol to the sample, vortexed well, transferred to miTotal ^TM RNA Column and centrifuged for one minute, centrifuged down the liquid discarded, and added 500 μl WS Buffer, centrifuged one minute After the liquid was poured off, the action was repeated and the WS buffer was added twice in total. After centrifugation for three minutes, spun down to ensure that the liquid completely, the miTotal ^TM RNA Column moved to the new RNA enzyme-free centrifuge tube. Add 50μl of the enzyme RNA-free water to the miTotal ^TM RNA Column, centrifuged at full speed for one minute, i.e., to obtain a micro RNA (of microRNA) and total RNA (totalRNA). Please save this sample at minus 70 degrees immediately. The result is shown in Figure 5.

Those skilled in the art should be aware that changes can be made to the above examples without departing from the broad inventive concepts. Therefore, it is to be understood that the invention is not limited to the specific examples of the present invention, and is intended to cover the modifications of the invention and the scope of the invention as defined by the appended claims.

The foregoing summary of the invention, as well as the above detailed description For the purposes of illustration of the present invention, various drawings are illustrated in the drawings. However, it should be understood that the invention is not limited to the precise arrangements and devices disclosed.

In each of the drawings: FIG. 1 is a microRNA and a total RNA which can be extracted into osteosarcoma cells according to the present invention and compared with a conventional method for extracting RNA, which can obtain a large yield and The quality is better; FIG. 2 is a microRNA and total RNA extracted by the method according to another example of the present invention, and can be compared with the traditional method for extracting RNA, and the method can be compared. Good quality; 3 is a microRNA and total RNA extracted from spleen tissue and liver tissue according to another example of the present invention; FIG. 4 is an extract of gold dew in accordance with another example of the present invention; MicroRNAs and total RNAs and compared with traditional methods for extracting RNA, this method can obtain larger yield and better quality; FIG. 5 is a method for extracting according to another example of the present invention. To the tobacco microRNA and total RNA, and compared with the traditional method of extracting RNA, this method can obtain better quality.

Claims (8)

A method for extracting microRNAs and total RNAs, comprising: (a) a decomposition solution (b) a centrifugation tube The method for extracting microRNAs and total RNAs according to claim 1 of the patent scope, wherein the decomposition solution covers phenol (Phenol) and derivatives. The method for extracting microRNAs and total RNAs according to claim 1 of the patent scope, wherein the decomposition solution covers Guanidine Thiocyanate and derivatives. For example, the method for extracting microRNAs and total RNAs of claim 1 wherein the pH of the decomposition solution covers pH 4-6. For example, in the method of extracting microRNAs and total RNAs of claim 1, wherein the decomposition solution is thoroughly mixed with the sample, and isopropanol is added before the tube is centrifuged. And its derivatives or ethanol and its derivatives. For example, the proportion of sample mixtures in the scope of patent application is 30%-70%. The method for extracting microRNAs and total RNAs according to claim 1 of the patent scope, wherein the centrifugal operation tube material Contains plastic, acrylic or metal of iron, aluminum, copper or alloy. The method for extracting microRNAs and total RNAs according to claim 1, wherein the filter layer material of the centrifuge tube comprises glass fibers, foams, non-woven fabrics or sieves.