Sterilization of pipette tips and EP tubes, etc.

1. Prepare 0.1% (one thousandth) DEPC (highly toxic substance) with deionized water, use it carefully in a fume hood, and store it at 4°C away from light;

DEPC water is pure water treated with DEPC and sterilized by high temperature and high pressure. Tested to be free of RNase, DNase and proteinase.

2. Put the pipette tip and EP tube into 0.1% DEPC, and ensure that the pipette tip and EP tube are filled with 0.1% DEP.

3. Protect from light, let stand, overnight (12-24h)

4. The box containing the tip and EP tube does not need to be soaked in DEPC. After roughly removing the DEPC water in the tip or EP tube, pack it up and wrap it up.

5. 121 degrees Celsius, 30min

6. 180 degrees Celsius, dry for several hours (at least 3 hours)

Note: a. Wear latex gloves and masks when handling DEPC! b, or without DEPC sterilization, 130 ℃, 90min autoclave (many laboratories high temperature sterilization twice)

RNA extraction considerations

Two major phenomena of tissue RNA isolation failure

RNA degradation and residues of impurities in tissues, regarding degradation, let’s first look at why RNA extracted from cultured cells is not easily degraded. Existing RNA extraction reagents all contain components that rapidly inhibit RNase. Add the lysate to the cultured cells, and simply mix it, all the cells can be thoroughly mixed with the lysate, and the cells are completely lysed. After the cells are lysed, the active ingredients in the lysate immediately inhibit the intracellular RNase, so the RNA remains intact. That is to say, because the cultured cells are easily and fully contacted with the lysate, their RNA is not easily degraded; on the other hand, the RNA in the tissue is easily degraded because the cells in the tissue are not easy to quickly contact the lysate. due to sufficient contact. So, assuming there is a way to turn the tissue into a single cell while inhibiting RNA activity, the problem of degradation could be completely solved.

Liquid nitrogen milling is the most effective such method. However, the liquid nitrogen milling method is very troublesome, especially when the number of samples is large. This gave rise to the next best thing: the homogenizer. The homogenizer method does not consider the question of how RNase activity is inhibited before cells are contacted with the lysate, but rather prays that the rate of tissue disruption is faster than the rate at which intracellular RNase degrades RN.

The effect of electric homogenizer is better, and the effect of glass homogenizer is poor, but in general, the homogenizer method cannot prevent the degradation phenomenon. Therefore, if the extraction is degraded, the original electric homogenizer should be used for grinding with liquid nitrogen; the original glass homogenizer should be changed to an electric homogenizer or directly milled with liquid nitrogen. The problem is almost 100% feasible. get resolved.

The impurity residue problem affecting subsequent experiments has more diverse causes than degradation, and the solutions are correspondingly different. In conclusion, if there is degradation or residual impurities in the tissue, the extraction method/reagent for the specific experimental material must be optimized. You don’t have to use your precious samples for optimization: you can buy some small animals like fish/chicken from the market, take the corresponding part of the material for RNA extraction, and the other part for protein extraction – grind with mouth, stomach and intestines Extract.

The target RNA of the extracted RNA is used for different follow-up experiments, and its quality requirements are different

cDNA library construction requires RNA integrity without residues of enzyme reaction inhibitors; Northern requires higher RNA integrity and lower requirements for enzyme reaction inhibitors residues; RT-PCR does not require too high RNA integrity, but inhibits enzyme reactions. Residue requirements are strict. The input determines the output; every time the goal is to obtain the highest purity RNA, it will cost the people and money.

Collection/Storage of Samples

Factors Affecting Degradation After the sample leaves the living body/or the original growth environment, the endogenous enzymes in the sample will begin to degrade RNA, and the degradation rate is related to the content of endogenous enzymes and temperature. Traditionally, there are only two ways to completely inhibit endogenous enzyme activity: add lysate immediately and homogenize thoroughly and rapidly; cut into small pieces and immediately freeze in liquid nitrogen. Both approaches require fast operation. The latter is suitable for all samples, while the former is only suitable for tissues with low content of cells and endogenous enzymes and easier to homogenize. Specifically, plant tissue, liver, thymus, pancreas, spleen, brain, fat, muscle tissue, etc. are best frozen with liquid nitrogen before proceeding.

Fragmentation and homogenization of samples

Factors Affecting Degradation and Yield Sample fragmentation is for thorough homogenization, which is for complete and complete release of RNA. Cells can be directly homogenized without being broken. Tissues can be homogenized only after being broken. Yeast and bacteria need to be broken with corresponding enzymes before they can be homogenized. Tissues with lower endogenous enzyme content and easier homogenization can be crushed and homogenized at one time in the lysate by a homogenizer; plant tissue, liver, thymus, pancreas, spleen, brain, fat, muscle tissue and other samples, They are either high in endogenous enzymes or are not easily homogenized, so tissue disruption and homogenization must be performed separately. The most reliable and most productive method of fragmentation is milling with liquid nitrogen, and the most reliable method of homogenization is the use of an electric homogenizer. A special note about milling with liquid nitrogen: the sample must not be thawed during the entire milling process, as endogenous enzymes are more likely to function when frozen.

Choice of lysate

Affecting the convenience of operation and the factors of residual endogenous impurities The commonly used lysis solutions can almost inhibit the activity of RNase. Therefore, the key point of choosing a lysis solution is to consider in combination with the purification method. There is one exception: samples with high endogenous enzyme content are recommended to use a lysate containing phenol to increase the ability to inactivate endogenous enzymes.

Choice of purification method

Factors that affect residual endogenous impurities, extraction speed For clean samples such as cells, satisfactory results can be obtained with almost any purification method at hand. But for many other samples, especially those with high levels of impurities such as plants, liver, bacteria, etc., choosing a suitable purification method is crucial. The column centrifugal purification method has a fast extraction speed and can effectively remove impurities that affect the subsequent enzymatic reaction of RNA, but it is expensive(Foregene can offer cost-effective kits, more details click here); using economical and classic purification methods, such as LiCl precipitation, can also obtain satisfactory results, but the operation time is long. .

“Three Disciplines and Eight Attentions” for RNA Extraction

Discipline 1: Put an end to the contamination of exogenous enzymes.

Note 1: Strictly wear masks and gloves.

Note 2: The centrifuge tubes, Tip heads, pipette rods, electrophoresis tanks, and experimental benches involved in the experiment should be thoroughly disposed of.

Note 3: The reagents/solutions involved in the experiment, especially water, must be RNase-free.

Discipline 2: Block the activity of endogenous enzymes

Note 4: Choose an appropriate homogenization method.

Note 5: Choose an appropriate lysate.

Note 6: Control the starting amount of the sample.

Discipline 3: Clarify your extraction purpose

Note 7: With any lysate system approaching the maximum starting amount of sample, the extraction success rate drops sharply.

Note 8: The only economic criterion for successful RNA extraction is a success in subsequent experiments, not yield.

Top 10 Sources of RNase Contamination

1. Fingers are the first source of exogenous enzymes, so gloves must be worn and replaced frequently. In addition, masks must also be worn, because breathing is also an important source of enzymes. An additional benefit of wearing a glove mask is to protect the experimenter.

2. Pipette tips, centrifuge tubes, pipettes – RNase cannot be inactivated by sterilization alone, so pipette tips and centrifuge tubes should be treated with DEPC, even if they are marked as DEPC treated. It is best to use a special-purpose pipette, wipe it with a 75% alcohol cotton ball before use, especially the rod; in addition, be sure not to use a head remover.

3. The water/buffer must be free of RNase contamination.

4. At least the test table should be wiped clean with 75% alcohol cotton balls.

5.Endogenous RNase All tissues contain endogenous enzymes, so quick freezing of tissues with liquid nitrogen is the best way to reduce degradation. The liquid nitrogen storage/grinding method is indeed inconvenient, but it is the only way for tissues with high levels of endogenous enzymes.

6. RNA samples RNA extraction products may contain traces of RNase contamination.

7. Plasmid extraction Plasmid extraction often uses Rnase to degrade RNA, and the residual Rnase should be digested with Proteinase K and extracted by PCI.

8. RNA storage Even if it is stored at low temperature, trace amounts of RNase will cause RNA degradation. The best solution for long-term preservation of RNA is a salt/alcohol suspension, because alcohol inhibits all enzymatic activity at low temperatures.

9. When cations (Ca, Mg) contain these ions, heating at 80C for 5 minutes will cause RNA to be cleaved, so if RNA needs to be heated, the preservation solution needs to contain a chelating agent (1mM Sodium Citrate, pH 6.4).

10. Enzymes used in subsequent experiments may be contaminated by RNase.

10 Tips for RNA Extraction

1: Quickly prevent RNase activity. Samples are quickly frozen after collection, and RNase is inactivated by rapid operation during lysis.

2: Choose an appropriate extraction method for tissue with high ribozyme content, and adipose tissue is best to use the method containing phenol.

3: Prediction quality requires Northern, cDNA library construction requires high integrity, and RT-PCR and RPA (Ribonuclease protection assay) do not require high integrity. RT-PCR requires high purity (enzyme inhibitor residues).

4: Thorough homogenization is the key to improving yield and reducing degradation.

5: Check the integrity of RNA electrophoresis detection, 28S: 18S = 2: 1 is a complete sign, 1: 1 is also acceptable for most experiments.

6: Removal of DNA for RT-PCR, array analysis It is best to use Dnase I to remove DNA.

7: Reduce the contamination of exogenous enzymes – enzymes cannot be imported from the outside.

8: When concentrating low-concentration nucleic acid, a co-precipitation reagent should be added. But to prevent the co-precipitant containing enzymes and DNA contamination.

9: Thoroughly dissolve the RNA, if necessary, heat at 65C for 5 minutes.

suitable storage method

It can be stored at –20C for a short time, and at –80C for a long time. The first step in improving RNA yields is to realize that the RNA content of different samples varies greatly. High abundance (2-4ug/mg) such as liver, pancreas, heart, medium abundance (0.05-2ug/mg) such as brain, embryo, kidney, lung, thymus, ovary, low abundance (<0.05ug/mg) mg) such as bladder, bone, fat.

1: Lyse cells to release RN – if RNA is not released, the yield will be reduced. Electric homogenization works better than other homogenization methods, but may also need to be combined with other methods, such as liquid nitrogen mashing, enzymatic digestion (Lysozyme/Lyticase)

2: Optimization of the extraction method. The biggest problems with phenol-based methods are incomplete stratification and partial RNA loss (the supernatant cannot be completely removed). Incomplete stratification is due to high nucleic acid and protein content, which can be solved by increasing the amount of lysate used or reducing the amount of sample. A step of chloroform extraction was added to the adipose tissue. RNA loss can be reduced by back-pumping or by removing the organic layer followed by centrifugation. The biggest problem with column centrifugation-based methods is excess sample.

Classic Extraction Tips

1. Phenol purification: Add an equal volume of 1:1 Phenol/Chloroform and mix vigorously for 1-2 minutes. Centrifuge at high speed for 2 minutes. Carefully remove the supernatant (80-90%). Never get to the middle layer. An equal volume of the reaction solution can be added to Phenol/Chloroform and the supernatant removed. The two supernatants can be mixed together for nucleic acid precipitation to improve the yield. Don’t be too gentle when mixing, and don’t try to remove all the supernatant.

2. Washing with 70-80% ethanol: During washing, the nucleic acid must be suspended to ensure that the residual salt is washed away. At the same time, immediately after pouring off the ethanol, centrifuge at high speed for a few seconds, and then remove the residual ethanol with a pipette. Dissolve after standing at room temperature for 5-10 minutes.

11. Extraction of special organizations

1. Fibrous tissue: The key to RNA extraction from fibrous tissue such as heart/skeletal muscle is to completely disrupt the tissue. These tissues have low cell density, so the amount of RNA per unit weight of tissue is low, and it is best to use as much starting amount as possible. Be sure to grind the tissue thoroughly under freezing conditions.

2. Tissues with high protein/fat content: brain/vegetable fat content is high. After PCI extraction, the supernatant contains white floccules. The supernatant must be re-extracted with chloroform.

3. Tissues with high nucleic acid/ribozyme content: the spleen/thymus has high nucleic acid and ribozyme content. Grinding tissue under freezing conditions followed by rapid homogenization can effectively inactivate ribozymes. However, if the lysate is too viscous (due to high nucleic acid content), the PCI extraction will not be able to stratify effectively; adding more lysate can resolve this issue. Multiple PCI extractions can remove more residual DNA. If a white precipitate forms immediately after adding alcohol, it indicates DNA contamination. Re-extraction with acidic PCI after dissolution can remove DNA contamination.

4. Plant tissue: Plant tissue is more complex than animal tissue. Generally, plants are ground under liquid nitrogen conditions, so RNA degradation by endogenous enzymes is uncommon. If the degradation problem is not resolved, it is almost certainly caused by impurities contained in the sample. The impurities contained in many plants will lead to residues, and the reason for residues is often because these impurities have some similarities with RNA: you precipitate and I precipitate, and you adsorb and I adsorb. These characteristics determine that they are very strong enzyme inhibitors.

At present, commercial RNA extraction reagents can be adapted to almost all animal tissues with small adjustments, but there are few commercial RNA extraction reagents that can be suitable for most plant tissues. Fortunately, Foregene can provide special plant RNA extraction kits, we have Plant Total RNA Isolation kit, Plant Total RNA Isolation kit Plus. The latter is specially designed for plants with high polysaccharide and polyphenol content. For RNA extraction, feedback from lab users is particularly good.

12. The effect of sample freezing and thawing The frozen sample may be larger, and it needs to be cut before being used for RNA extraction. Samples tend to melt (possibly partially) during cutting. Frozen samples may need to be weighed before RNA extraction, and thawing will definitely occur during this process. Sometimes, the thawing of the sample also occurs during the liquid nitrogen milling process; or the frozen sample is directly added to the lysate without liquid nitrogen milling, and thawing will definitely occur before the complete homogenization. Experiments have shown that frozen tissue is more prone to RNA degradation during thawing than fresh tissue. The likely reason: The freeze-thaw process disrupts structures within the cell, making it easier for endogenous enzymes to come into direct contact with the RNA.

13. Judgement of RNA quality Usually, electrophoresis is used to judge the integrity of RNA, and A260/A280 is used to judge the purity of RNA. In theory, intact RNA has a ratio of 28S:18S = 2.7:1, and most data emphasize the ratio of 28S:18S = 2:1. The fact is that almost none of the RNA extracted from samples other than cells is in a 2:1 ratio (this was obtained using the Agilent Bioanalyzer).

The electrophoresis results of RNA are affected by many factors, including secondary structure, electrophoresis conditions, sample load, degree of saturation by EB, etc. Use native electrophoresis to detect RNA and use DNA Marker as a control. If the 28S at 2kb and the 18S at 0.9kb are clear, and 28S: 18S > 1, the integrity can meet the requirements of most subsequent experiments.

A260/A280 is an indicator that has caused a lot of confusion. First of all, it is necessary to clarify the original meaning of this indicator for nucleic acids: pure RNA, its A260/280 = about 2.0. Pure RNA is the ’cause’ and A260/A280 = 2 is the ‘effect’. Now everyone is using A260/A280 as a ’cause’, thinking that “if A260/A280 = 2, then RNA is pure”, which naturally leads to confusion.

If you are interested, you can add a little reagent that is often used in extraction, such as phenol, guanidine isothiocyanate, PEG, etc., to your RNA sample, and then measure the A260/A280 ratio. The reality is that many of the reagents used for RNA extraction, as well as many impurities in the sample, absorb around A260 and A280, affecting A260/A280.

The most instructive approach at present is to scan RNA samples in the 200-300 nm range. The curve of pure RNA has the following characteristics: the curve is smooth, A230 and A260 are two inflection points, A300 is close to 0, A260/A280 = around 2.0, and A260/A230 = around 2.0. If scan data are not available, the A260/A230 ratio must also be determined, as this ratio is more sensitive to carryover of all impurities that affect the enzymatic reaction. Take into account the linear range of the device (0.1–0.5 for A260).

There are two other useful phenomena: the ratio will be about 0.3 lower when A260/A280 is measured in water; while the ratio measured in 10 mM EDTA is about 0.2 higher than that measured in 1 mM EDTA.

Related products:

China Plant Total RNA Isolation Kit Manufacturer and Supplier | Foregene (foreivd.com)

RNA isolation series Suppliers and Factory | China RNA isolation series Manufacturers (foreivd.com)

RNA isolation series – Foregene Co., Ltd. (foreivd.com)