DNA or genetic material includes the encoded genetic blueprint of a cell. In order to get the information about the entry of DNA into cytoplasm, as it never leaves the nucleus of the cell, where it can meet other biochemical elements and proteins, it is essential to convert DNA into RNA, a messenger which includes polyRNA or mRNA, at first. Then this mRNA is converted into proteins to perform a number of functions of that cell. In order to know about very rare mRNAs you will have to create libraries of corresponding molecules of DNA or investigate for microarrays, which makes it necessary to separate mRNA from the total RNA. But the process of the extraction of total RNA and the process of isolating resultant mRNA cannot be performed exclusively. So to know how to separate mRNA from total RNA? You should read this write-up till end.
Separation of mRNA from total RNA
mRNA can be separated from total RNA through various methods including:
Homogenization with Trizol: The total RNA includes transfer RNA, ribosomal RNA, mRNA and other RNAs that are non-coded. In order to separate all the RNAs from each other the cell has to release its contents by burst opening it by suspending the cells shot by spinning them at high speed in the reagent, Trizol or its other versions.
Isolation of total RNA: In order to separate the components like DNA, RNA and proteins of a cell into phases or layers within the suspension a series of centrifugations are used. Its top phase yellow in color can be discarded because it is made of fat. The second phase tinted red in color is retained as a desired phase as it contains total RNA. This total RNA can be shot to separate mRNA by performing a series of washes by using ethanol and isopropanol and extraction of phenol-chloroform. In order to prevent degrading of total RNA the formation of enzyme RNase is slowed down by adding inhibitors. You can get total RNA here.
Extraction of mRNA: Normally a kit is used to extract highly purified mRNAs which may not be possible by using homemade lab rules. Some basis steps used by these kits for extracting mRNA from total RNA may include:
- Mix almost 300 micro-liters of total RNA with the RNase-inhibited buffer lysis provided in the kit
- Heat the mixture at 65 degree C for 5 minutes and cool its sample instantly for a minute on ice
- Now mix the cooled sample with 0.5M Sodium Chloride and add Oligo dT or oligodeoxythymidylic acid in to the mixture to dissolve completely
- In order to recover supernatant the sample is then centrifuged which is then washed with low salt and various binding buffers provided with the kit several times.
- Now mRNA can be eluted by washing the supernatant several times with some solvent until the desired amount of mRNA is not received.
- This elute is then precipitated by using precipitates of ethanol and sodium acetate. The resultant is then re-suspended in nearly 20 micro-liters water treated by diethylpyrocarbonate or DEPC.