Efficient disruption and homogenization of the starting material is an essential requirement for all total RNA isolation operations. Fragmentation and homogenization are two separate steps.

• Fragmentation: Complete disruption of tissue structure, cell wall and plasma membrane is absolutely necessary to release all RNA in the sample. Different samples require different methods to achieve a thorough crushing effect. Incomplete fragmentation results in a significant reduction in RNA yield.
• Homogenization: Homogenization is necessary to reduce the viscosity of cell lysates after fragmentation. Homogenization can cleave high molecular weight genomic DNA and other high molecular weight cellular components to obtain a uniform cleavage product. Insufficient homogenization results in a decrease in RNA binding efficiency and a significant decrease in yield.

Some crushing methods can homogenize the sample at the same time, while other methods require a special homogenization step. The criteria for crushing and homogenizing different samples provide a comprehensive overview of the different crushing and homogenizing methods applicable to a variety of starting materials. This form can be used as a reference when you choose the appropriate method of operation for the starting material used. The crushing and homogenizing methods are also discussed in more detail below.

Crushing and homogenizing using a bead mill

During the process of crushing the sample using a bead mill, the sample and the beads were stirred at high speed. The crushing and homogenization process is completed simultaneously by hydrodynamic cutting and crushing when the beads collide with the cells. The factors affecting the crushing efficiency are:

• The size and composition of the beads
• Buffer to bead ratio
• Number of starting samples
• Mixing speed and setting
• Processing time

The ideal bead type used for bacterial disruption is 0.1 mm (average diameter) glass beads, 0.5 mm glass beads for yeast and single cell animal cells, and 3–7 mm stainless steel beads for animal and plant tissues. . Make sure the glass beads are pre-washed with concentrated nitric acid. Alternatively, commercially available acid washed glass beads can be purchased and used directly. Other parameters regarding crushing need to be set according to the experience of each application. Plant material and associated beads and crushing tubes can be pre-cooled in liquid nitrogen, and the disruption should be carried out without lysis buffer. For animal tissue, dry freeze comminution should be used. Freezing and comminution (whether in a bead mill or using a mortar and pestle) is different from buffer lysis and does not simultaneously homogenize the sample.

Crushing and homogenizing using a rotor-stator homogenizer

In the presence of lysis buffer, the rotor-stator homogenizer can simultaneously complete the complete fragmentation and homogenization of animal tissue, based on the toughness of the sample, which can be completed in 5–90 seconds. The rotor-stator homogenizer can also be used for the homogenization of cell lysates. The ultra-high-speed rotation of the rotor enables the crushing and homogenization of the sample in a comprehensive manner of disturbance and mechanical shearing. Using a suitably sized tube, the homogenizer head is always submerged during the homogenization process and the homogenizer head is continuously extended into the end of the tube to ensure minimal foam in the sample during homogenization. The rotor-stator homogenizer is available in different sizes and can be fitted with probes of different sizes. The 5 mm and 7 mm probes are suitable for use in volumes below 300 μl and can be used for homogenization in centrifuge tubes. Probes of 10 mm and larger are suitable for larger tubes.

Use mortar and pestle to break

When using a mortar and pestle, the sample is first subjected to rapid liquid nitrogen freezing and ground to a fine powder in liquid nitrogen. Transfer the supernatant (tissue powder and liquid nitrogen) to a suitably sized tube pre-cooled with liquid nitrogen to allow liquid nitrogen to evaporate but do not allow the sample to melt. Add lysis buffer and proceed as quickly as possible.

Note: Grinding a sample with a mortar and pestle will break the sample but will not achieve homogenization. The homogenization needs to be carried out separately from this step.

Use a syringe and needle to complete the homogenization

The lysate of cells and tissues can be homogenized using a syringe and a needle. A sterile plastic syringe can be attached using a 20 gauge (0.9 mm) needle and the high molecular weight DNA is cleaved by at least 5–10 blows until the homogenate of the lysate is formed. For ease of handling and reduced sample loss, it may also be necessary to increase the volume of the lysis buffer.

Some sample sources differ significantly in their RNA or inclusions and may cause problems in the isolation and analysis of RNA. Some special considerations are required when operating these sample sources. This section will explore the operation of a large number of different sample sources.

plant

The isolation of RNA from plant material encounters some particular difficulties, and conventional techniques are generally conditionally optimized prior to use in plant samples. Some plant metabolites are chemically similar to nucleic acids, making them difficult to remove from RNA preparation products. Co-segregation of metabolites (eg, polysaccharides, polyphenols, and flavonoids) or contaminants with the target RNA by improperly used purification methods (such as salts or phenol) may result in inhibition of enzymatic reactions Or lead to deviations in UV spectrophotometry and gel electrophoresis results. Difficulties that may be encountered in the process of isolating RNA from plant material include volumetric errors due to higher viscosity and RNA degradation during storage.

Plant growth conditions are usually optimized so that they do not produce high levels of plant metabolites and can contribute to efficient separation of RNA. Due to the large differences between plant species, it is difficult to have the same guiding criteria for their growth conditions. However, as a general principle, it is recommended to use healthy and young plant tissues whenever possible. The RNA yield of young plant tissues is usually higher than that of older tissues because the former usually contains more cells than the latter under equal weight conditions. And equal weight younger tissues usually contain fewer metabolites. In addition, many "custom" RNA isolation methods recommend harvesting plant tissue in the dark for 1-2 days before harvesting to avoid the formation of high levels of plant metabolite accumulation.

Heart, muscle and skin tissue

Isolation of RNA from samples such as skeletal muscle, heart, and skin tissue can be difficult because such samples contain large amounts of contractile proteins, connective tissue, and collagen. In order to remove these proteins that may interfere with RNA isolation, such samples need to be treated with proteases or phenol-containing lysis reagents. However, the digestion process of proteases needs to avoid degradation of RNA.

bacterial

Many of the basic features of bacterial mRNA are different from those of eukaryotes. Prokaryotic mRNA does not have a 5' cap and rarely has a poly A tail. mRNA lacking a poly A tail cannot be captured by hybridization. In addition, it is not possible to use oligo-dT primers in the synthesis of the initial strand, and only random primers can be used instead.

In addition, bacterial RNA is also very unstable, and the average half-life of RNA in rapidly growing varieties is about 3 minutes. The mRNA of some bacteria is degraded at the same time as translation. So for researchers trying to isolate mRNA from bacteria, this can be a big problem. It is also due to the fact that the turnover of mRNA in bacteria (from generation to degradation) is very fast, making gene expression studies in prokaryotes more difficult than eukaryotes. In order to accurately preserve the gene expression profile and maximize the yield of intact mRNA, it is necessary to stabilize the sample before it is taken and processed.

blood

The blood is directly derived from the collected samples for clinical analysis. RNA from blood samples can be successfully saved using RNA stabilization reagents in collection tubes. Methods for isolating RNA from blood require the ability to ensure the production of high quality RNA without contaminants or enzyme inhibitors. The large amount of enzyme inhibitors contained in the blood can interfere with downstream RNA analysis. In addition, a class of conventional anticoagulants such as heparin and EDTA can interfere with downstream analysis. It should be noted that anticoagulants do not stabilize RNA.

Red blood cells (red blood cells) in human blood do not contain nuclei and therefore do not synthesize RNA; in general, such cells contain only very small amounts of RNA, not the main target of RNA isolation. The primary target cells for RNA isolation from whole blood are white blood cells (white blood cells), which contain nuclei and RNA. White blood cells are composed of three major cell types: lymphocytes, monocytes, and granulocytes.

Since healthy blood contains about 1000 times more red blood cells than white blood cells, removing red blood cells can help simplify the RNA separation step. This can be achieved by selectively lysing red blood cells, since red blood cells are more sensitive to hypotonic shock (compared to white blood cells) and can rupture rapidly in hypotonic buffers.

Another common alternative to lysing red blood cells is Ficoll density-gradient centrifugation. Unlike the erythrocyte lysis method, Ficoll density-gradient centrifugation only acquires mononuclear cells (lymphocytes and monocytes), but removes granulocytes. Monocytes isolated by Ficoll density gradient centrifugation can be used for RNA isolation in the same manner as other animal cells.

FFPE tissue sample

Formalin-fixed, paraffin-embedded (FFPE) tissue samples are an important and extensive source of samples for biomedical research. More and more researchers are beginning to analyze molecular levels at the beginning of FFPE samples, so it is increasingly important to consider the unique properties of these samples to develop targeted separation methods.

Due to the use of formaldehyde during immobilization and embedding, severe fragmentation and chemical changes in the nucleic acids in the FFPE sample are typically caused. Therefore, the nucleic acid isolated from the FFPE sample will have a lower molecular weight than the fresh or frozen sample. The degree of fragmentation varies based on the type of sample, the length of the shelf life, and the conditions of fixation, embedding, and storage. Although formaldehyde modification cannot be measured in standard quality control analyses such as gel electrophoresis or lab-on-a-chip analysis, the latter actually causes significant interference with enzymatic analysis.

In order to minimize the impact of FFPE storage on RNA transcripts, the following tips should be kept in mind:

• Sampling and fixing tissue as quickly as possible
• Do not use samples with a thickness of more than 5 mm. The sample fixing time should not be too long (up to 24 hours)
• Use high quality reagents for paraffin embedding, do not add other additives
• Avoid staining samples as much as possible
• Store FFPE samples appropriately (RNA can be stored at 4 °C for 1 year, better than room temperature or higher)
• Use the appropriate deparaffinization step
• RNA isolation should include a step to decrosslink

Pure RNA can be stored in RNase-free water at –20 °C or –70 °C. Under the above conditions, RNA degradation does not occur within 1 year.

The above information is from the official website of QIAGEN:

Http://