[Rui Sai's small subject] Extraction and electrophoresis of total RNA from miRNA research animals

I. Experimental purpose and requirements
A. Understand and master the technical principles and methods of operation for extracting and purifying total RNA from animals.
B. Understand and master the technical principle and operation method of identifying the total RNA of the obtained animal by electrophoresis.

II. Experimental principle and background knowledge
A. RNA is a biological macromolecule that is important for gene expression in life activities, such as mRNA, which carries all the encoded information of DNA. Isolation of RNA is one of the important foundations for studying gene function and plays an important role in molecular biology. The extracted mRNA can be used in experiments such as Northern blot, RT-PCR, construction of cDNA libraries, Gene Chips, and in vitro translation.
B. In this test, since RNase is extremely stable, it is necessary to be careful when handling RNA to prevent RNA from being degraded. The RNase inactivation must be performed on the vessel used prior to RNA extraction. Treat glassware with 180oC dry baking for more than 8 hours, or soak glassware and other supplies with 0.1% aqueous solution of diethylpyrocarbonate (DEPC). The water used and the relevant buffer also need to be treated with 0.1% DEPC water, but Tris-Cl buffer or the like cannot be treated with DEPC. (Note: DEPC is suspected of causing cancer and must be handled with care to prevent contact and inhalation!)
C. In animal cells, the RNA contained is mainly rRNA (80 to 85%), tRNA and small RNA (10 to 15%) and mRNA (1 to 5%). The rRNA content is the most abundant, consisting of 28S, 18S and 5S. There are many kinds 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) layer can be used according to this property. The column separates the mRNA from the total RNA. In general, the extracted total RNA can also be used in Northern blot experiments.
D. The following problems should be noted in the experimental operation of extracting animal RNA:
1. Generally, the animal tissue cells are broken by mechanical grinding or homogenization;
2. To add a protein denaturant to separate the nucleoprotein from the RNA and release the RNA;
3. Inhibition of endogenous and exogenous RNase activities;
4. Separate RNA from DNA, proteins, and other cellular components.
E. Overall, there are currently more applications in the experiment, and there are three methods for extracting total RNA:
1. Phenol method: using SDS to denature protein and inhibit RNase activity, after removing phenol/chloroform to remove protein, polysaccharide, pigment, etc., precipitating RNA with NaAC and ethanol;
2. Strontium salt method: denatured protein with guanidinium isothiocyanate or guanidine hydrochloride and b-mercaptoethanol, and inhibited the activity of RNase, and then precipitated after chloroform extraction;
3. Lithium chloride precipitation method: Since lithium can precipitate RNA relatively specifically at a certain pH, it is easy to lose small RNA, and residual lithium ions have an inhibitory effect on mRNA.
F. In this experiment, the RNA extraction process was performed using Invitrogen's TRIzol reagent, the principle of which is based on the sulfonium salt method. That is, the animal tissue powder is lysed in an extract containing a strong guanidinium isothiocyanate denaturing agent, and an RNase inhibitor is contained in the extraction buffer to inhibit RNase activity and ensure RNA integrity. The sample can be fully cleaved in the TRIzol reagent. After centrifugation with chloroform, the solution forms a supernatant layer, an intermediate layer and an organic layer (lower layer). The RNA is distributed in the supernatant layer, and the supernatant layer is collected and then subjected to isopropanol. Total RNA can be recovered by precipitation.
G. Electrophoresis and detection of total RNA from the extracted animals:
The detection of RNA is mainly carried out by agarose gel electrophoresis, which is divided into non-denaturing electrophoresis and denaturing electrophoresis. The most common denaturing electrophoresis is formaldehyde denaturing electrophoresis (as in the Northern blot experiment).
Since the RNA molecule is a single-stranded nucleic acid molecule, which 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 obtain dependence on the conventional agar pond gel electrophoresis. The molecular weight electrophoresis separation strip, for which the sample is heat denatured at 65 degrees Celsius for 5 minutes before the electrophoresis is applied, the secondary structure of the RNA molecule is fully opened, and an appropriate amount of formaldehyde is added to the agarose gel to ensure that the RNA molecule is The single-stranded state is maintained during the electrophoresis process. Therefore, the total RAN sample obtains a molecular weight-dependent, stepwise separation band on the agarose gel in a uniform conformation. Moreover, the denaturation of the RNA facilitates the binding to the nitrocellulose membrane during the transfer process. RNA can be analyzed visually and rapidly by formaldehyde-denaturing agarose gel electrophoresis. When a standard "molecular scale" is present, the total RNA sample can be qualitatively and quantitatively determined. In order to determine the size of the fragment, the label can be electrophoresed on the same piece of glue, and then the gel is cut, colored, and photographed.

Sharing is a virtue, then share it.
Colon cancer fund mature subject, responsive
Search the public number "Rui Sai Bio" to choose to pay attention
The phone scans the QR code below to follow

On the WeChat client, you can click on “Rui Sai Classroom” in the “Rice Race Activity” at the bottom to see more exciting information.
Ruisai Bio R&S  
There is fresh technology information here, and there are biological creatures here.
There is a scientific research sentiment here, and everything here is related to biology.
Oncology articles, department articles, experimental techniques articles, information sections, submissions:

Plastic Hinges

Plastic Hinges,Plastic Door Hinges,Small Cabinet Hinges,Plastic Cabinet Hinges

Ningbo Hengchieh Locking Technology Co., Ltd. , https://www.hengchieh.com