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Extraction of whole blood DNA and RNA
How to choice the suitable methods for the extraction of whole blood DNA and RNA?
It is a basic work for extracion DNA and RNA from whole blood, whose quality affects the downstream experiments and analysis directly. There are many methods to be chosen, which confuse us, so it is difficult for many people to choice the most suitable method before the experiment. Now we will analyze the most suitable method for the experiment from two sides step by step.
1 Selection and Preservation of whole blood and extraction of DNA
a Take blood using blood collection tube with anticoagulants which is EDTA or heparin generally, but EDTA is better that would not affect the downstream experiments. If blood collection tube is not available, anticoagulant EDTA could be added into blood derectly and mixed fully, in which every 0.4ml EDTA solution per 5 ml blood that 0.04M EDTA solution shoul be prepared in advance. It could be generally extracted within 2-3 years when it is stored at -20℃ to -80℃, but the storage time is shorter and the quality of extraction is higher, so the best way is extracting DNA within 2 months.
b, Choice of DNA extraction method: There are two types in Whole blood extraction kit, spin-column type and solution type, (eg. in Qiagen, Promega and Bioteke, the phonel chloroform method is abandoned) whose principles and procedures are the same. Although many models of DNA extraction kit was introduced by many company that people do not know how to choose, basically the DNA extraction kit contains solution and spin-column two types from the principles and procedures. Generally speaking, the purity of DNA extracted by spin-column(DP1801) is higher, but the capacity is smaller(0.1-1ml) and the yield is lower slightly. While the capacity of solution type(DP2101 or DP 2201) is larger(1-10ml), and the yield is higher than spin-column. In general, the blood sample from hospitals is above 2ml, and the solution type is chosen for it. Refered to the differences of products between Chinese and other countries’, many people think that the goods from other countries must be better than from China, but what is the trueth? Only by trying to know, besides the price of goods from China is much closer to people. The genomic DNA extraction kit (DP2101 and DP2201) creat the third-generation technology without Protease K digestion after constantly optimizing the experiments, while it is need digested by Protease K when using the kit from other countries. Both DP 2101 and DP2201 could reduce the trouble and time of preparation of Protease K solution, and the quality of extraction and stability are much better.(The products use pure chemical methods to lyse the cell, so do not worry about lysing instability cause by the activity of enzyme.)
c, Can non-anticoagulation blood(coagulated blood) be extracted? The answer is affirmative, but also no bother extraction, and there is no difference to anticoagulated blood! There are few products for coagulated blood DNA extraction, however, the coagulated blood DNA extraction kit from Bioteke has had a lot of loyal user.
2, The extraction of whole blood RNA
a, Which are the factors in RNA degradation during extracting RNA from whole blood?
RNase is abundant in whole blood, so RNA is easy to degradation in the absence of protection! What situations of RNA unprotected are the process of lysing red blood cells by erythrocyte lysate which is a low concentration of salt solution and do not contain inhibition ingredients of RNase, and the process of digestion by DNase during which there is no inhibition ingredients of RNase.
b, What is a better method of extraction?
We tend to select the method that RNA is protect in the entire process. It is not the best method that lysing the erythrocyte with erythrocyte lysis buffer at first, and nucleic acid is extracted from the leucocyte, then digesting DNA with DNase, during which there are a lot of factors for RNA degradation. Therefore, direct lysis method is the best way to extract RNA, in which three times the volume of buffer RLS (the lysis buffer in RP4001 Blood/Liquid Sample Total RNA Rapid Extraction kit(Spin-column))is added into the seleced blood, and reversed to mix fully. Lysis buffer RLS contains strong protein denaturant that can rapidly make protein denaturated, also did RNase, so that it can protect RNA from degradation. The mixture after lysing can also be used for transport, in order to avoid RNA degradation.
|blood , dna , dna rna extraction , extraction , rna|