I. Experimental purpose and requirements
A. Understand and master the technical principles and operation methods of extracting and purifying total animal RNA
B. Understand and master the technical principles and operation methods of total animal RNA extracted by electrophoresis identification.
II. Experimental principles and background knowledge
A. RNA is a biological macromolecule that is very important 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 the study of gene function and 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, since RNase is extremely stable, you should take extra care when handling RNA to prevent RNA from being degraded. Before extracting RNA, the vessels used must be inactivated by RNase. For example, use 180oC dry baking for more than 8 hours to treat glassware, or soak glassware and other supplies with 0.1% diethyl pyrocarbonate (DEPC) in water. The water and related buffers also need to be treated with 0.1% DEPC water, but Tris-Cl buffers, etc. cannot be treated with DEPC. (Note: DEPC is suspected to be carcinogenic and must be handled carefully to prevent contact and inhalation!)
C. In animal cells, the RNA contained is mainly rRNA (80-85%), tRNA and small molecule RNA (10-15%) and mRNA (1-5%). rRNA is the most abundant and consists of 28S, 18S and 5S. There are many types of mRNAs, with molecular weights ranging from hundreds to thousands of bases, but most mRNAs have a Poly-A tail at the 3 'end. Therefore, oligodeoxythymidine (Oligo dT) layers can be used according to this characteristic The separation column separates the mRNA from the total RNA. In general, the total RNA extracted 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. The animal tissue cells are generally broken by mechanical grinding or homogenization;
2. To add protein denaturant to separate nucleoprotein and RNA and release RNA;
3. Inhibit endogenous and exogenous RNase activity;
4. Separate RNA from DNA, protein and other cellular components.
E. In general, there are many applications in the experiment. There are three mature methods for extracting total RNA:
1. Phenol method: Denature protein with SDS and inhibit RNase activity. After multiple phenol / chloroform extractions to remove proteins, polysaccharides, pigments, etc., use NaAC and ethanol to precipitate RNA;
2. Guanidine salt method: denature the protein with guanidine isothiocyanate or guanidine hydrochloride and b-mercaptoethanol, and inhibit the activity of RNase, and then precipitate after extraction with chloroform;
3. Lithium chloride precipitation method: because lithium can precipitate RNA relatively specifically at a certain pH, but it is easy to cause the loss of small-molecule RNA, and the residual lithium ion has an inhibitory effect on mRNA.
F. In this experiment, the RNA extraction process used Invitrogen's TRIzol reagent, the principle of which was based on the guanidine salt method. That is, the animal tissue powder is lysed in an extract containing a strong guanidine isothiocyanate denaturant, and the RNase inhibitor is contained in the extraction buffer to inhibit RNase activity and ensure the integrity of RNA. The sample can be fully lysed in TRIzol reagent. After adding chloroform and centrifuging, the solution will form a supernatant layer, an intermediate layer and an organic layer (lower layer). The RNA is distributed in the supernatant layer. The total RNA can be recovered by precipitation.
G. Electrophoresis and detection of total animal RNA extracted:
The detection of RNA is mainly carried out by agarose gel electrophoresis, which is divided into non-denaturing electrophoresis and denaturing electrophoresis. The most commonly used denaturing electrophoresis is formaldehyde denaturing electrophoresis (such as during Northern blot experiments).
Because the RNA molecule is a single-stranded nucleic acid molecule, it is different from the double-stranded molecular structure of DNA. It can be folded back to form a hairpin secondary structure and a more complex molecular state, so that it is difficult to rely on the traditional agar pond gel electrophoresis. Separate bands of molecular weight for electrophoresis. For this purpose, heat and denature the sample at 65 degrees Celsius for 5 minutes 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 has a stepwise separation band on the agarose gel that depends on the molecular weight in a uniform conformation. Moreover, after denaturation of RNA, it is beneficial to the combination with nitrocellulose membrane during the transfer process. RNA can be analyzed intuitively and quickly by formaldehyde denaturing agarose gel electrophoresis. When there is a standard "molecular ruler" (marker), the total RNA sample can be qualitatively and quantitatively objectively quantified. In order to determine the size of the fragment, the same gel can be electrophoresed with a marker, and then the gel can be cut off and applied.
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