Product Information 

Product Description

       This kit uses a special centrifugal adsorption column and buffer system to quickly extract total RNA from animal tissues/cells. The extraction can be completed in about 25 minutes, and the operation is simple and easy to use. The extracted total RNA does not contain protein and other impurities, and has extremely high purity. It can be used for RT-PCR, Real Time RT-PCR, chip analysis, Northern Blot, Dot Blot, Poly A screening, in vitro translation, molecular cloning and other downstream experiments.

      This animal tissue/cell RNA rapid extraction kit has strong versatility and good compatibility, and can be widely used in the extraction of various tissue/cell samples. It has been verified that it can be used to extract NIH/3T3 cells, HeLa cells, COS-7 cells, LMH cells, Huh cells, mouse or rat embryos, kidneys, livers, spleens, lungs, thymus and other cell or tissue samples.

Product composition

Features

1. No toxic reagents such as phenol and chloroform are used, and no steps such as ethanol precipitation are required.

2. Simple, single sample operation can generally be completed within 25 minutes.

3. It has a wide range of adaptability and can be used to extract various cell or tissue samples including NIH/3T3 cells, HeLa cells, COS-7 cells, LMH cells, Huh cells, mouse or rat embryos, kidneys, livers, spleens, lungs, thymus, etc.

4. Multiple column washing ensures high purity. The typical ratio of OD ₂₆₀ / OD ₂₈₀ is as high as 2.1-2.2. There is basically no DNA residue. It can be used for RT-PCR, Northern-blot and various experiments.

Transportation and storage methods

1. This kit can be stored at room temperature for 12 months without affecting its effectiveness.

2. Storing the test kit at low temperature (4℃ or -20℃) will cause solution precipitation and affect the use effect. Therefore, transportation and storage should be carried out at room temperature (15℃-25℃).

3. Avoid long-term exposure of reagents to air to prevent volatilization, oxidation, and pH changes. Close the lid of each solution immediately after use.

Typical cell/tissue RNA yields

RNA content in different cells or tissues

Precautions

1. The centrifuge used in the experiment can be operated at room temperature (4°C is also acceptable) and the speed can reach 12,000rpm.

2. You need to prepare ethanol, β-mercaptoethanol or DTT and mortar.

3. The sample processing volume cannot exceed the processing capacity of the centrifugal column, otherwise it will easily lead to DNA residue or reduced RNA yield.

4. RLT and RW1 contain irritating compounds. Be sure to wear gloves when operating to avoid contamination of skin, eyes and clothes. If contaminated with skin or eyes, rinse with plenty of water or saline.

5. The sample processing volume should not exceed the processing capacity of the adsorption column, otherwise it will cause DNA residue or reduce the yield. The RNA/DNA of different tissue cell types varies greatly. For example, the thymus and spleen are rich in DNA content, and if it exceeds 5mg, it will exceed the column processing capacity. COS cells are rich in RNA content, and if it exceeds 3x106 ^cells  , it will exceed the column adsorption capacity. Therefore, when you start to explore the experimental conditions, if you are not sure about the sample DNA/RNA content, you would rather use a smaller sample processing volume, such as cells not exceeding 3-4×106 ^and tissues not exceeding 10mg. Increase or decrease the processing volume according to the sample test results later.

6. About trace amounts of DNA residues:

Generally speaking, any total RNA extraction reagent cannot completely avoid trace amounts of DNA residue during the extraction process (DNase digestion cannot achieve 100% residue-free). Our RNA extraction products, due to the use of our company's special lysis solution system and centrifuge tube adsorption column technology, have removed most of the DNA and do not require DNase digestion, and can be directly used for RT-PCR and qPCR. In some special cases, such as excessive DNA content causing residues or strict mRNA expression analysis by fluorescence quantitative PCR, we recommend the following when selecting templates and primers:

(1) Select intron-spanning primers that cross the junction region in the mRNA so that the DNA cannot serve as a template in the amplification reaction.

(2) Select primer pairs that produce products of different sizes on genomic DNA and cDNA.

(3) Before rinsing with deproteinizing buffer RW1 in step 1, perform DNase I on-column digestion directly on the centrifuge column. You can ask for specific operating instructions before purchasing the DNase I on-column digestion kit (Cat. No.: HRN0291).

7. RNA purity and concentration detection:

Integrity: RNA integrity can be tested by ordinary agarose gel electrophoresis (electrophoresis conditions: gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15 minutes). Since 70%-80% of the RNA in cells is rRNA, very obvious rRNA bands should be visible under UV after electrophoresis. The sizes of animal rRNA are approximately 5 kb and 2 kb, which are equivalent to 28S and 18S rRNA, respectively. The brightness of the maximum rRNA in the animal RNA sample should be 1.5-2.0 times the brightness of the second largest rRNA, otherwise it indicates degradation of the RNA sample. The appearance of diffuse flakes or the disappearance of bands indicates that the sample is severely degraded.

Purity: The OD260/OD280 ratio is a reference indicator for measuring the degree of protein contamination. For high-quality RNA, the OD260/OD280 reading (10mM Tris, pH7.5) is between 1.8-2.1. The OD260/OD280 reading is affected by the pH value of the solution used for the measurement. For the same RNA sample, assuming that the OD260/OD280 reading measured in 10mM Tris, pH7.5 solution is between 1.8-2.1,

In aqueous solution the reading may be between 1.5 and 1.9, but this does not mean that the RNA is impure.

Concentration: Take a certain amount of RNA extract, dilute it n times with RNase-free water, and use RNase-free water to measure the concentration of RNA in the spectrophotometer.

Adjust to zero, take the diluted solution for OD260 and OD280 determination, and calculate the RNA concentration according to the following formula: final concentration (ng/μL) = (OD260)×(dilution factor n)×40.

8. This product is for scientific research purposes only!

How to use

hint:

→ Before first use, please add the specified amount of anhydrous ethanol to Buffer RPE and Buffer RW1 and mark them!

→ Add 10 μL of β-mercaptoethanol or 20 μL of 2M DTT to 1 mL of Buffer RLT before use. Lysis buffer containing β-mercaptoethanol or DTT can be stored at room temperature for up to 1 month.

1. Tissue culture cells

1. Collect less than 107 ^suspended cells into a 1.5mL centrifuge tube. For adherent cells, cells cultured in well plates can be directly lysed. Cells cultured in cell culture flasks should be digested with trypsin first and then collected by blowing.

2. Centrifuge at 12,000 rpm for 10 seconds to precipitate the cells. Discard the supernatant completely, leaving the cell pellet. Note that incompletely discarding the supernatant will dilute the lysate, resulting in reduced yield and purity.

3. Gently tap the tube wall to completely loosen the cell pellet and resuspend it. Add 350μL (<5×10 ⁶ cells) or 600μL (5×10 ⁶ -1×10 ⁷ cells) of lysis buffer RLT. Mix by pipetting and shake vigorously by hand for 20 seconds to fully lyse the cells.

4. Homogenization: (When the number of cells to be treated is very small (less than 1×10 ⁵⁾ , it is generally not necessary to vortex and oscillate for 1 minute to homogenize). Use a 1 mL disposable syringe with a blunt needle (with a 0.9 mm needle) to vigorously pump the lysate for more than 10 times or until a satisfactory homogenization result is obtained (or electric homogenization for 30 seconds). This can shear DNA, reduce viscosity to prevent column clogging and increase content.

5. Add the entire lysis mixture or homogenate mixture to the gDNA cleanup column (the cleanup column is placed in the collection tube).

6. Immediately centrifuge at 12,000 rpm for 60 seconds and retain the filtrate (RNA is in the filtrate).

7. Use a micropipette to estimate the volume of the filtrate more accurately (usually 350μL/600μL, the volume lost during filtration should be subtracted), add an equal volume of 70% ethanol, precipitation may occur at this time, but it does not affect the extraction process, immediately blow and mix, do not centrifuge. Immediately add the mixture (less than 700μL each time, can be added twice at most) to the RNA adsorption column, put the adsorption column into the collection tube, immediately centrifuge at 12,000rpm for 30 seconds, and discard the waste liquid.

8. Add 700 μL Buffer RW1, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

9. Add 500 μL Buffer RPE (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, add 500 μL Buffer RPE again, and repeat.

10. Place the RNA adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 min. Remove the rinse solution as much as possible to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction.

11. Remove the RNA adsorption column and place it in a new enzyme-free centrifuge tube. Add 30-50 μL of enzyme-free water to the middle part of the adsorption membrane, place it at room temperature for 1 minute, and centrifuge it at 12,000 rpm for 1 minute. The liquid obtained in the centrifuge tube is RNA.

If the expected RNA yield is >30 μg, add 30-50 μL of enzyme-free water and repeat step 12. Combine the two eluates, or use the first eluate and add it back to the adsorption column and repeat the step (if a high RNA concentration is required).

The RNA elution solution for two elutions has a higher concentration. The RNA yield of the combined elution solution from two elutions is 15-30% higher than the former, but the concentration is lower.

2. Animal tissues (e.g. mouse liver and brain)

1. Electric homogenization: Cut fresh tissue into small pieces quickly with a scalpel, add 350μL (<20mg tissue) or 600μL (20-30mg tissue) of lysis buffer Bufffer RLT and homogenize thoroughly with an electric honiton for 20-40 seconds.

2. Grinding and homogenization in liquid nitrogen: Grind the tissue into powder in liquid nitrogen, take an appropriate amount of tissue powder (20mg/30mg) and transfer it into a 1.5mL centrifuge tube containing 350μL/600μL tissue lysis buffer Bufffer RLT, shake it vigorously by hand for 20 seconds to fully lyse. Use a disposable 1mL syringe with a blunt needle (with a 0.9mm needle) to vigorously pump the lysate 10 times or until a satisfactory homogenization result is obtained (or electrophoresis).

Homogenize for 30 seconds) to shear DNA, reduce viscosity, prevent column clogging and increase yield.

3. Centrifuge the homogenized lysate at 12,000 rpm for 3 min to precipitate any fragments or insoluble matter that may be difficult to lyse, and add all the supernatant of the lysate to the DNA adsorption column (the adsorption column is assembled in the collection tube).

4. Immediately centrifuge at 12,000 rpm for 60 seconds and save the filtrate (RNA is in the filtrate).

5. Use a micropipette to more accurately estimate the volume of the filtrate (usually 350μL/600μL, the volume lost during filtration should be subtracted), add an equal volume of 70% ethanol, precipitation may occur at this time, but it will not affect the extraction process, immediately blow and mix, do not centrifuge, immediately add the mixture (less than 700μL each time, can be added twice at most) to the RNA adsorption column, put the adsorption column into the collection tube, immediately centrifuge at 12,000rpm for 30 seconds, and discard the waste liquid.

6. Add 700 μL Buffer RW1, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

7. Add 500μL Buffer RPE (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000rpm for 30 seconds, discard the waste liquid, add 500μL Buffer RPE again, and repeat.

8. Place the RNA adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 min. Remove as much rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions.

9. Remove the RNA adsorption column and place it in a new enzyme-free centrifuge tube. Add 30-50 μL of enzyme-free water to the middle part of the adsorption membrane, place it at room temperature for 1 minute, and centrifuge it at 12,000 rpm for 1 minute. The liquid obtained in the centrifuge tube is RNA.

If the expected RNA yield is >30 μg, add 30-50 μL of enzyme-free water and repeat step 12. Combine the two eluates, or use the first eluate and add it back to the adsorption column and repeat the step (if a high RNA concentration is required).

The RNA elution solution from two elutions has a higher concentration. The RNA yield from the combined elution solution from two elutions is 15-30% higher than the former, but the concentration is lower.

Appendix 1: Table of cell numbers in adherent culture