Nucleic Acid Extraction Instrument

TriZol Total RNA Extraction Reagent

Product Item Number:R1000

Storage conditions:TriZol Total RNA Extraction ReagentCan be stably stored at room temperature for 12 months.We suggest saving it inIn an environment of 2-8 ° C.

Important Notice:

This product contains phenol, which is toxic and corrosive. If inhaled, in contact with the skin, swallowed, etc., it can cause poisoning, burns, and other bodily injuries. When using this product, protective equipment such as gloves, eye masks, face shields, etc. should be worn. If accidentally touched, immediately rinse with plenty of water and seek medical treatment.

Product Introduction

TriZol Total RNA Extraction ReagentIt is a broad-spectrum total RNA extraction reagent. The experimental operation is fast and convenient, with bright colors and easy layering. This reagent has a wide range of applications and can extract total RNA from animal tissues, plant materials, various microorganisms, and cultured cells. This method has good separation effects on small amounts of tissues (50-100mg) and cells (5 × 106), as well as large amounts of tissues (≥ 1g) and cells (>107). The sample was fully lysed in TriZol total RNA extraction reagent while ensuring RNA integrity as much as possible. After centrifugation with chloroform, the solution will be divided into three layers: the upper colorless aqueous phase, the middle layer, and the lower red organic phase, with RNA distributed in the supernatant layer. After collecting the supernatant, total RNA can be recovered by precipitation with isopropanol. The extracted total RNA has good integrity, no protein or DNA contamination, and can be used for various routine molecular biology experiments, such as RT-PCR、Real-time RT-PCR、Northern blot、Dot Blot、 External translation, etc.

The TriZol total RNA extraction reagent can promote the precipitation of multiple RNAs of different species and molecular weights. For example, RNA agarose gel electrophoresis extracted from rat liver and stained with ethidium bromide showed many discontinuous high molecular weight bands (mRNA and hnRNA components) between 7 kb and 15 kb, two dominant ribosomes~5 kb (28S) and~2 kb (18S), and low molecular weight RNA between 0.1 and 0.3 kb (tRNA, 5S). When the extracted RNA is diluted with TE, its A260/A280 ratio is ≥ 1.8. Note that for ordinary agarose gel electrophoresis, the position of 28S is about 2kb, and 18S is about 1kb. The position of gel with different concentrations varies greatly.

Notes:

1.for sample useAfter homogenizing the TriZol total RNA extraction reagent, if chloroform is not added immediately, it can be stored at -70 ° C for more than a month. RNA precipitation stored in 75% ethanol can be stored for one week at 2-8 ° C and for one year at -20 ° C. RNA has a relatively short half-life and is easily degraded. It is recommended to conduct subsequent experiments as soon as possible after extraction, such as reverse transcription into cDNA, Northern Blot, etc.

If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with RNase free DNase I.

3.Self prepared reagents: chloroform, isopropanol（Newly opened or extractedRNA specific), 75% ethanol (prepared with DEPC treated water), RNase free water, or DEPC treated water.

4. Our company produces TriZol Total RNA Extraction Reagent with excellent quality, which can replace Invitrogen's Trizol Reagent. The extraction quality is the same as downstream experiments.

RNA Extraction Procedure: (Please read the precautions before the experiment)

Hint

useWear gloves and eye protection when extracting RNA with TriZol total RNA extraction reagent. Avoid contact with skin and clothing. Complete the operation in the chemical fume hood. Avoid inhalation through the respiratory tract. Unless otherwise specified, all operations should be carried out at room temperature of 15-30 ° C.

1. Homogenate

a. Plant tissue:

Take fresh plant tissue and grind it thoroughly in liquid nitrogen or crush it directly after processingTriZol Total RNA Extraction ReagentGrind rapidly, add 1ml TriZol total RNA extraction reagent to every 50-100mg tissue, and mix well. Note: The sample volume should generally not exceed 10% of the total RNA extraction reagent volume of TriZol.

b. Animal organization:

Fresh or-Cut animal tissues frozen at 70 ℃ as much as possible, add 1ml TriZol total RNA extraction reagent to every 30-100mg tissue, and homogenize with a homogenizer. Alternatively, after grinding in liquid nitrogen, add 1ml of TriZol total RNA extraction reagent and mix well. Note: The sample volume should generally not exceed 10% of the total RNA extraction reagent volume of TriZol.

c. Single layer cultured cells:

Try to remove any residual culture medium as much as possible and directly increase the diameterAdd 1ml of TriZol total RNA extraction reagent to a 3.5cm culture plate, cover and repeatedly blow and lyse the cells. Determine the required amount of TriZol total RNA extraction reagent based on the area of the culture plate rather than the number of cells (1ml per 10cm2). When the total RNA extraction reagent of TriZol is insufficient, it can lead to DNA contamination in the extracted RNA.

Note:

Wall attached cultured cells often cannot detach from the culture bottle (dish), which does not mean that lysis is not possible. At this point, the cell membrane has actually ruptured and released all of itRNA， Just keep doing it.

d. Cell suspension:

Collect cells by centrifugation. inTriZol Total RNA Extraction Reagent: Use a pipette to repeatedly blow and lyse cells. Every 5-10 times10⁶Add 1ml of animal cells, plant or yeast cells, or 1ml of bacteria per 1 × 107 cellsTriZol Total RNA Extraction ReagentWash cells should be avoided before adding TriZol total RNA extraction reagent, as it increases the possibility of mRNA degradation. Cracking certain yeast and bacteria may require the use of a homogenizer.

f. Blood:

useTriZol Total RNA Extraction ReagentReagent LS。

2.Shake the homogenized sample vigorously and place it at room temperature5 minutes to dissociate the nuclear protein body.

3. Optional steps:

inUnder the condition of 4 ° C and a centrifugal force of 12000 rpmCentrifuge for 10 minutes and take the supernatant.

If the sample contains a high amount of protein, fat, polysaccharides or muscle, plant tubers and nodules can be removed by centrifugation. The centrifuged precipitate contains cell outer membrane, polysaccharides, and high molecular weightDNA， The supernatant contains RNA. When processing samples of adipose tissue, a large amount of oil should be removed from the upper layer. Take the clarified homogenate for the next step.

4.each1mlTriZol Total RNA Extraction ReagentAdd 0.2ml chloroform. Cover the tube tightly, shake vigorously for 15 seconds, and let it sit at room temperature for 2-3 minutes.

5.inHigh speed freeze centrifugation at 4 ° C and 12000 rpm for 10-15 minutes. After centrifugation, the mixture is divided into three layers: the lower layer is a red organic phenol chloroform layer, the middle layer, and the upper layer is a colorless water sample layer. RNA is present in the aqueous layer without exception. The capacity of the water sample layer is approximately 50-60% of the total RNA extraction reagent capacity of TriZol added. The organic layer and intermediate layer consist of protein and DNA. If extraction is required, please contact us for the extraction method.

6.Transfer the water sample layer to a clean centrifuge tube and add an equal volume of isopropanol. After reversing and mixing, let it stand at room temperature10 minutes.

RNA precipitation is usually not visible before centrifugation, but forms a gel like precipitate on the tube side and bottom after centrifugation.

7.At room temperature orCentrifuge at 12000 rpm at 4 ° C for 10 minutes and discard the supernatant.

8.joinWash the precipitate with 75% ethanol. Wash the precipitate with 1 ml of 75% ethanol for every 1 ml of TriZol total RNA extraction reagent used.

9.At room temperature orCentrifuge at 12000 rpm at 4 ° C for 3 minutes, discard the supernatant, and be careful not to lose the RNA precipitate.

Attention: The remaining small amount of liquid can be briefly centrifuged and then sucked out with a gun tip, being careful not to discard the sediment.

10.Room temperature storage2-3 minutes, air dry. Add 30-100 μ l RNase free water, dissolve RNA thoroughly, and store the obtained RNA at -70 ℃ to prevent degradation.

Attention: The remaining small amount of liquid can be briefly centrifuged and then sucked out with a gun tip, being careful not to discard the sediment.