I. Experiment purpose and requirements

　　A. Understand and master the technical principles and operating methods of extracting and purifying total animal RNA.

　　B. Understand and master the technical principles and operating methods of total animal RNA extracted by electrophoresis.

　　II. Experimental principles and background knowledge

　　A. RNA is a very important biological macromolecule in the process of gene expression in life activities, such as mRNA, which carries all the coding information of DNA. The isolation of RNA is one of the important foundations for studying gene function, and it occupies an important position in molecular biology. The extracted mRNA can be used in Northern blot, RT-PCR, cDNA library construction, Gene Chips, and in vitro translation experiments.

　　B. In this experiment, because RNase is extremely stable, you must be extra careful when handling RNA to prevent RNA from being degraded. Before extracting RNA, the utensils used must be inactivated by RNase. For example, dry baking at 180oC for more than 8 hours to treat glassware, or soak glassware and other supplies with 0.1% diethyl pyrocarbonate (DEPC) aqueous solution. The water used and related buffers also need to be treated with 0.1% DEPC water, but Tris-Cl buffers, etc. cannot be treated with DEPC. (Caution: DEPC is suspected of causing cancer, it must be handled carefully to prevent contact and inhalation!)

　　C. In animal cells, the RNA contained is mainly rRNA (80-85%), tRNA, small RNA (10-15%), and mRNA (1-5%). The rRNA content is the most abundant and consists of 28S, 18S and 5S. There are many types of mRNA, ranging in molecular weight from hundreds to thousands of bases, but most mRNAs have a Poly-A tail at the 3'end. Therefore, oligodeoxythymidine (Oligo dT) layer can be used according to this feature The separation column separates mRNA from total RNA. In general, the extracted total RNA can also be used in Northern blot experiments.

　　D. The following points should be paid attention to in the experimental operation of extracting animal RNA:

　　1. Generally use mechanical grinding or homogenization to break animal tissue cells;

　　2. Add protein denaturant to separate nucleoprotein from RNA and release RNA;

　　3. Inhibit endogenous and exogenous RNase activity;

　　4. Separate RNA from DNA, proteins and other cellular components.

　　E. In general, there are currently more applications in experiments, and there are 3 more mature methods for extracting total RNA:

　　1. Phenol method: Use SDS to denature protein and inhibit RNase activity. After multiple phenol/chloroform extractions to remove protein, polysaccharides, pigments, etc., RNA is precipitated with NaAC and ethanol;

　　2. Guanidine salt method: denature protein with guanidine isothiocyanate or guanidine hydrochloride and b-mercaptoethanol, and inhibit the activity of RNase, then re-precipitate after chloroform extraction;

　　3. Lithium chloride precipitation method: Because lithium can precipitate RNA relatively specifically at a certain pH, it is easy to cause the loss of small molecule RNA, and the residual lithium ions have an inhibitory effect on mRNA.

　　F. In this experiment, Invitrogen's TRIzol reagent was used in the RNA extraction process. The principle is based on the guanidine salt method. That is, the animal tissue powder is lysed in the extract containing strong guanidine isothiocyanate denaturant, and the extraction buffer contains RNase inhibitor to inhibit RNase activity and ensure the integrity of RNA. The sample can be fully lysed in TRIzol reagent. After adding chloroform and centrifugation, the solution will form a supernatant layer, an intermediate layer and an organic layer (lower layer). RNA is distributed in the supernatant layer. After the supernatant layer is collected, it is subjected to isopropanol The total RNA can be recovered by precipitation.

　　G. Electrophoresis and detection of extracted animal total RNA:

　　The detection of RNA mainly uses agarose gel electrophoresis, which is divided into non-denaturing electrophoresis and denaturing electrophoresis. General denaturing electrophoresis is most commonly used for formaldehyde denaturing electrophoresis (such as during Northern blot experiments).

　　Because RNA molecule is a single-stranded nucleic acid molecule, it is different from the double-stranded molecular structure of DNA. It can fold back to form a hairpin secondary structure and a more complex molecular state, so that it is difficult to rely on traditional agar pond gel electrophoresis. Molecular weight electrophoresis separates bands. For this purpose, heat and denature the sample at 65 degrees Celsius for 5 minutes before electrophoresis loading to fully open the secondary structure of the RNA molecule, and add an appropriate amount of formaldehyde to the agarose gel to ensure that the RNA molecule is in The single-stranded state is continuously maintained during the electrophoresis process. Therefore, the total RAN sample obtains the molecular weight-dependent stepwise separation bands on the agarose gel in a uniform configuration. Moreover, the denaturation of RNA is beneficial to the combination with the nitrocellulose membrane during the transfer process. RNA through formaldehyde denaturation agarose gel electrophoresis can directly and quickly analyze the quality of RNA. When there is a standard "molecular marker" (marker), the total RNA sample can be qualitatively and quantified relatively objectively. In order to determine the size of the fragments, the same gel can be electrophoresed with markers, and then the gel is cut, colored and photographed.