Ribonuclease (RNase) is a very stable, active enzyme that usually functions without the need for cofactors. Since ribonuclease is difficult to inactivate, and a small amount is sufficient to destroy RNA molecules, do not use any plastic or glassware without first eliminating any possible ribonuclease contamination. Care should be taken when handling RNA to avoid unintentional introduction of ribonuclease into the RNA sample during or after purification. In order to create and maintain an environment free of ribonucleases, the following precautions must be taken when pretreating and processing RNA using disposable and non-disposable vessels and solutions.

Routine operation

When handling RNA, be sure to use appropriate microbiological aseptic techniques. Hand and dust particles can carry bacteria and mold and are the most common source of ribonuclease contamination. In order to prevent ribonuclease contamination from dust on the skin surface or laboratory equipment, latex gloves or vinyl gloves (PVC gloves) are always worn when handling reagents and RNA samples. If possible, always change gloves and turn the tube off. When pipetting the solution for downstream applications, the purified RNA is placed on ice.

To remove RNase contamination on bench surfaces, non-disposable plastic containers, and laboratory instruments (eg, pipettes and electrophoresis tanks), it is recommended to use a commercially available ribonuclease removal solution. It is also possible to remove ribonuclease contamination using common laboratory reagents. To remove contamination on plastic vessels, wash with 0.1 M NaOH, 1 mM EDTA, and then with RNase-removed water; if the plastic vessel is resistant to chloroform, it can be washed with chloroform. To remove contamination from the electrophoresis tank, detergent cleaning (eg, 0.5% SDS) can be used, washed with RNase-containing water, then with ethanol (if the electrophoresis tank is resistant to ethanol), and then air dried.

IMPORTANT: When using chemical reagents, be sure to wear appropriate lab coats, disposable gloves, and goggles. Please refer to the corresponding Safety Data Sheet (SDS) provided by the product manufacturer for more information.

Disposable plastic consumables

When handling RNA, it is recommended to use sterile, disposable polypropylene tubing. These tubes are usually free of ribonucleases and therefore do not require pretreatment to inactivate ribonuclease.

Non-disposable plastic consumables

Non-disposable plastic consumables should be treated prior to use to ensure they do not contain ribonuclease. Plastic consumables should be washed strictly with 0.1 M NaOH, 1 mM EDTA, and then with RNase-free water. Plastic consumables with chloroform resistance can be washed with chloroform to inactivate ribonuclease.

glassware

Glassware should be treated to ensure that it does not contain ribonuclease before use. Glassware used to treat RNA should be cleaned with detergent, rigorously washed, and baked in an oven at 240 ° C for at least 4 hours (if convenient, overnight). Glassware can also be treated with DEPC (diethyl pyrocarbonate). The glassware was filled with 0.1% DEPC (0.1% aqueous solution), overnight (12 hours) at 37 ° C, then treated with autoclave or heated to 100 ° C for 15 minutes to remove the remaining DEPC.

IMPORTANT: When using chemical reagents, be sure to wear appropriate lab coats, disposable gloves, and goggles. Please refer to the Safety Data Sheet (SDS) provided by the product manufacturer for more information.

Electrophoresis tank

The electrophoresis tank should be cleaned with a detergent (for example, 0.5% SDS), washed strictly with RNase water, then with ethanol, and then air dried.

IMPORTANT: When using chemical reagents, be sure to wear appropriate lab coats, disposable gloves, and goggles. Please refer to the corresponding Safety Data Sheet (SDS) provided by the product manufacturer for more information.

Important: Some of the plastics used in the electrophoresis tank are not resistant to alcohol and require careful inspection of the manufacturer's instructions.

Solution

The solution (water or other solution) should be treated with 0.1% DEPC. DEPC is a potent but non-absolute RNase inhibitor that works by covalently modifying RNase.

IMPORTANT: When using chemical reagents, be sure to wear appropriate lab coats, disposable gloves, and goggles. Please refer to the corresponding Safety Data Sheet (SDS) provided by the product manufacturer for more information.

The solution (water or other solution) should be treated with 0.1% DEPC. DEPC is a powerful but not absolute RNase inhibitor. DEPC at a concentration of 0.1% is often used to treat glass or plastic consumables to inactivate RNase on its surface or to prepare RNase-free solutions and water. DEPC inactivates RNase by covalent modification. Add 0.1 ml of DEPC to 100 ml of the solution to be treated and shake vigorously to allow DEPC to fully enter the solution. The solution was incubated for 12 hours at 37 °C. It was treated in an autoclave for 15 min to remove any traces of DEPC. DEPC reacts with primary amines and therefore cannot be used directly to treat Tris buffer. In the presence of Tris buffer, DEPC is very unstable and rapidly decomposes into ethanol and CO2. When preparing Tris buffer, water should be treated with DEPC and then dissolved in Tris to prepare a suitable buffer. Trace amounts of DEPC are modified by reaction with hydrazine residues in the RNA molecule by acetylation. In an extracellular environment, acetylated RNA molecules are translated with very low efficiency. However, unless a large number of purine residues are modified, the ability of these RNA molecules to hybridize to form a DNA:RNA or RNA:RNA hybrid will not be severely affected. The DEPC remaining on the solution or vessel must be treated in an autoclave or heated to 100 ° C for 15 min for removal.

IMPORTANT: When using chemical reagents, be sure to wear appropriate lab coats, disposable gloves, and goggles. Please refer to the corresponding Safety Data Sheet (SDS) provided by the product manufacturer for more information.

To ensure the accuracy of gene expression analysis, the RNA analyzed should be able to accurately reflect its in vivo expression in biological samples. However, in the process of sample manipulation and RNA isolation, RNA is easily changed to complicate the situation.

Once the biological sample is extracted, the RNA therein becomes extremely unstable. The human influences that occurred during the period were mainly divided into two types. Down-regulation of genes and enzymatic degradation of RNA can result in artificially or non-specifically reduced mRNA. At the same time, certain genes are induced to be expressed during the processing and processing of the sample. The combination of these two effects may cause a bias between the detected transcriptional profile and the real situation in the body.

Immediate stabilization of RNA expression profiles is a top priority in accurate gene expression analysis. Typically, samples for RNA analysis are quickly frozen in liquid nitrogen and stored at –80 °C until further processing. Product suppliers also offer stabilization reagents as an alternative to stabilizing RNA in biological samples. These suppliers offer integrated sample processing solutions (packages) including containers, stabilization reagents and preparation kits

RNA size and molecular weight

From reference 2. Converted to Nucleic Acid: RNA

RNA molecular weight and molar conversion

MW (sodium salt) of single-stranded RNA molecule = (number of bases) x (343 Daltons/base)

* The average length of mRNA is 1930 nucleotides.

The above information is from QIAGEN's official website: http://