How to choose the best method for extracting DNA and RNA extraction from whole blood?
Extracting DNA and RNA from whole blood should be considered the most basic task, and the quality of the extracted DNA and RNA directly affects downstream experiments and analysis. There are many methods to choose from, and how to choose the most suitable method has become the most difficult thing for many people to decide before starting experiments. We analyze the best experimental method from two aspects, layer by layer cocooning.
1. Collection and preservation of whole blood and DNA extraction
I. Use blood collection vessels containing Anticoagulant to collect blood. Anticoagulant generally includes EDTA and heparin. EDTA is better and will not affect the downstream reaction. If there is no blood collection vessel, you can also add Anticoagulant EDTA yourself. Prepare 0.04M EDTA solution in advance. Add 0.4ml of prepared EDTA solution every 5ml of blood and mix it upside down. Stored at -20 ℃ to -80 ℃, it can generally be extracted within 2-3 years. The shorter the storage time, the higher the quality of extraction, and it is best to extract within 2 months.
II Selection of DNA extraction methods: The whole blood extraction kit generally has two types: centrifugal column and solution (such as Qiagen, Promega and Bioteke; the hand-held method of phenol chloroform is abandoned, which is toxic for a long time). The principle and steps are basically the same. Although various companies have launched more than N models, making it difficult to choose, from the principle and operating steps, there are basically two types: centrifugal column and solution type. Generally speaking, the extraction purity of centrifugal column (DP1801) is higher, but the processing capacity is small (0.1-1ml) and the yield is slightly lower; The extraction volume of solution type (DP2101 or DP2201) is large (1-10ml) and the yield is also higher than that of centrifugal column. The blood samples in hospitals are generally above 2ml, and the solution type method is generally chosen for extraction. When it comes to the difference between domestically produced and imported blood samples, many people believe that imported blood samples are definitely better than domestically produced blood samples. We believe that there is no best, only better! After continuous experimental optimization, the whole blood genome DNA extraction kit DP2101 or DP2201 has pioneered the third generation technology, which does not require Proteinase K digestion. Imported products need Proteinase K! DP2101 or DP2201 reduces the trouble of Proteinase K preparation and digestion time, and has better extraction amount and stability.
III. Can non anticoagulant (coagulated blood) be extracted? The answer is yes, and it's not at all troublesome. The extraction effect is no different from anticoagulation! After you have searched all the imported brands but can't find them, when you look back, you will find that he is around. There are many loyal users of Biotec's coagulated blood genome DNA extraction kit.
2. How to Extract Whole Blood RNA
I. What are the factors of RNA degradation in the whole blood RNA extraction process?
RNA enzymes in whole blood are very abundant, and RNA is easily degraded in an unprotected state! What are the state RNAs that are not protected, such as the process of red blood cell lysis by red blood cell lysate. Red blood cell lysate is a low concentration equilibrium salt solution that does not inhibit RNase components, in which case RNA is not protected; RNA is not protected during DNA enzyme digestion, and there is no component that inhibits RNase at this time.
II What extraction method is better?
When choosing methods, we should lean towards methods that protect RNA throughout the entire process! The general method is to first lyse red blood cells with red blood cell lysate, then extract nucleic acid from white blood cells, and also undergo digestion by DNase to remove DNA. This is not the best method, as there are many factors involved in RNA degradation during the process. So the direct lysis method is the best method. Quickly add 3 times the volume of the lysate RLS-RP4001 Blood (Liquid Sample) Total RNA Rapid Extraction Kit (Centrifuge Column Type) lysate to the collected blood, and quickly invert and mix. The lysate RLS contains a strong protein denaturant that can quickly denature proteins, which is RNase denaturation and cannot degrade RNA. The mixed solution after lysis can also be used for transportation to avoid RNA degradation, which is known as the production of expression profile chip RN by Boao Bio
Breakthrough in DNA extraction of environmental microorganisms
Pesticides, herbicides, various pollutants, synthetic substances and other foreign substances have a significant impact on the diversity and population changes of environmental and soil microorganisms; The detection of whether Genetically modified crops have undergone gene transfer in soil, the safety evaluation of Genetically modified crops and other studies need to accurately extract high-quality DNA for detection!
The number of cultivable microorganisms in soil may not be as high as 0.3% of the total number of microorganisms. Conventional methods for extracting DNA are difficult to extract, and it is difficult to remove all humic acids from the soil. However, trace amounts of humic acids may lead to PCR failure.
At present, although commercially available kits can extract the DNA of microorganisms in soil, the use of glass or steel beads for fragmentation methods has led to severe genome fragmentation and affected DNA quality; Secondly, due to the need to purchase specialized crushers or oscillators for crushing, their usage costs will increase; Thirdly, in order to ensure DNA purity, its reagent kit adopts a purification method that includes glass milk and centrifugal adsorption columns in series, resulting in a large amount of DNA loss and low yield, making it difficult to truly reflect the diversity of microorganisms in the soil.