RNA Extraction
RNA extraction that results in high signal and low noise, is of paramount importance for gene expression profiling and other associated analytical techniques. The goal is to concurrently stabilize labile target molecules during the extraction process, and to achieve higher quality and higher yield of target molecules. Numerous technologies exist for this process; however, this process has barely changed over the years, and these technologies now suffer from a number of drawbacks, which make them less than desirable, due to contact processing, and heat damage to samples.
The Covaris AFA™ process is ideally suited to sample disruption (homogenization). Conversely, in a rotor/stator process (e.g., Polytron), shearing is limited to a particle size, which is defined by the distance between the blades. Bead beaters, and mortar-and-pestle also have shear-stress size limits. For example, a mortar-and-pestle grinding in liquid nitrogen produces a particle of ~100um. At this size the particle "powder" is not a molecule, hence strong chemicals are required to diffuse into the sample to complete the standard inefficient process.
Covaris AFA instruments, in conjunction with the CryoPrep pulverization system, sometimes deliver high yields of high quality nucleic acid samples. For example the technology can:
- disrupt & homogenize tissue to molecular size level (<10 micron particle size)
- release target RNA biomolecules
- True pulverization of difficult tissues: bone, skin corneal, muscle, plants...
- Automated, standardized process—highly reproducible
- DNA and RNA can be extracted simultaneously from the same sample
- Non-contact, no cross contamination , no clean-up
- Isothermal, no heat damage to RNA samples
- Non-contact, high recovery for small mass samples
- Robust design, ideal for batch process
- Fast and easy
- No beads—neither sample loss, nor heat damage
- Automation potential for higher throughput processing
Above: RNA extraction from breast tumor tissue, ~40 mg starting material
The Covaris process gives more than 15 times of increased RNA yield comparing to conventional tissue preparatory process...
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Different processes
- Polytron
- Bead-beater
- Mortar/pestle
- Covaris
Tissues
- Lung ~120mg/sample
- Brain ~300mg/sample
- Kidney ~300mg/sample
- Femur & Tibia - Whole bone (used only for Covaris and mortar and pestle technique)
Comments
Mortar/pestle bone samples completely degraded RNA
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Above: RNA extraction from various tissue samples, using different processes
Comparison performed by Genzyme.
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Partial list of difficult tissue types disrupted using Covaris
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heart
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lung
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sciatic nerve
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bone
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liver
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nasal tissue
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cartilage
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eyes
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brain
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testes
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knee
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plant tissue - seeds
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kidney
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zebra fish
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How Do You Use the System?
For hard or fibrous tissue, we recommend an initial cryopulverization method. Use of the Covaris CryoPrep™ system greatly facilitates this step as it both fractures extracellular matrices (e.g., connective tissue) and increases the surface area for the acoustic extraction more details here.
Aqueous RNA extraction buffer (e.g., a guanidinium isothiocyanate buffer) is added to a frozen tissue sample specimen with or without a CryoPrep pretreatment in a TissueTube. A lid is placed on the tube and the tube is then placed into the Covaris AFA instrument. The instrument is then activated and the acoustic homogenization initiated. Typical homogenization times are 30 to 60 seconds. A proven combination is using:
1. Snap-freeze—TissueTube™ for collection and rapid, thermal stabilization
2. Freeze-fracture—CryoPrep™ for rapid pulverization
3. AFA extraction—Covaris S2 for rapid acoustic disruption and chemical stabilization
This three-step process is also beneficial for the isolation of labile biomolecules (e.g., active proteosome complexes). Often the "extraction" buffer is the stabilizer for the target biomolecule.
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