RNA Extraction: The Basics
Obtaining high-quality RNA is the first and perhaps the most critical step in several fundamental molecular biology experiments. To attain success in RNA extraction, you’ll need to go through several important steps before and after the purification of the extracted RNA. This article provides you with basic information on RNA isolation and extraction.
Sample Collection and Disruption
Ongoing research into RNA analysis has pinpointed two points in the process of RNA extraction that can still be improved: the treatment and handling of tissues or cells prior to RNA extraction, and storage of the extracted RNA. Since most of the actual RNA extraction occurs in a strong denaturant that generally renders RNases inactive, the integrity of RNA is most at risk before and after the extraction process.
It’s crucial to find the most appropriate method of cell disruption to ensure success in RNA extraction and purification. Just as importantly, one has to find the best RNA extraction method to attain the purest and best quality of yield. Following is a comparison of some of the most popular extraction and purification approaches.
1. Guanidine Isothiocyanate Extraction with Lithium Chloride Precipitation – In many cases, this approach involves the simultaneous lysing of cells, extraction of cellular RNA, and denaturing of proteins. It is lauded for inactivating RNases quicker than acid phenol extraction. With this approach, RNA and DNA from proteins are not physically separated in just one step, and protein contamination has to be removed by chloroform treatment.
2. Acid Phenol Extraction and Alcohol Precipitation – This is among the least expensive RNA extraction methods. However, it can be a very long procedure and is known to be prone to DNA contamination. Furthermore, this approach tends to leave residual phenol in the sample, thus introducing errors into RNA quantification. It also does not inactivate RNases immediately.
3. TRIzol – This approach combines the first two approaches and therefore entails some of their individual advantages. The drawback is that the approach depends largely on pipetting skills to remove protein and DNA. More importantly, phenol and chloroform are potentially harmful reagents, so one has to be extra careful in handling them.
4. Commercially Available Kits – Automated RNA extraction systems such as those offered by Aurora Biomed are currently the most widely recommended. Although it is more expensive than other methods, it is also among the most efficient.
As you are well aware of, it is essential to prevent contamination both before and after the RNA extraction procedure is performed. You should therefore be aware of the most common sources of contamination.
1. DNA – Perform a PCR of a housekeeping gene and a portion of the RNA preparation in order to identify DNA contamination. Take note that removing DNA with lithium chloride re-precipitation delivers the best results, but using DNasel delivers quicker results. 2. Protein – The A260/A280 ratio should be 2.0 for the purest RNA samples. Measure the ratio by diluting the RNA sample in TE buffer. If contamination is identified, a chloroform cleaning and re-precipitation may be required. 3. Salt – The A260/A240 ratio should be 1.4 for the purest RNA samples. Isopropanol precipitation plus 70% ethanol wash should remove contamination effectively.
To ensure success in RNA extraction and purification, you should take note of all these basic information and strictly follow RNA isolation protocols.
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Aurora Biomed provides instruments for RNA extraction needs
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