Molecular cloning chapter 07.docx

1、Molecular cloning chapter 07Chapter 7 Extraction, Purification, and Analysis of mRNA from Eukaryotic CellsProtocol 1: Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction In this single-step technique, cells are homogenized in guanidnium thiocyanate

2、 and the RNA is purified from the lysate by extraction with phenol:chloroform at reduced pH. Many samples can be processed simultaneously and speedily. The yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7 g/ml starting tissue or 5-10 g/106 cells. IMPORTANT

3、: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 2: A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues This protocol, a variation of the method described in Chapter 7, Protocol 1 , involves lysis of cells in a monophasic s

4、olution of guanidine isothiocyanate and phenol. Addition of chloroform generates a second (organic) phase into which DNA and proteins are extracted, leaving RNA in the aqueous supernatant. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 g/mg start

5、ing tissue or 5-10 g/106 cells. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 3: Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography Chromatography on oligo(dT) columns is the preferred method for large-scale purification (25 g) of poly(A)+ RNA extr

6、acted from mammalian cells. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA. Because the method can be frustratingly slow, it is not recommended for purification of poly(A)+ RNA from multiple samples. For this purpose, batch elution ( Chapter 7,

7、Protocol 4) is the better choice. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 4: Selection of Poly(A)+ RNA by Batch Chromatography When many RNA samples are to be processed or when working with small amounts (50 g) of total mammalian RNA, the technique of cho

8、ice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 5: Separation of

9、 RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels Separation of RNAs according to size is the first stage in northern blotting and hybridization. The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electr

10、ophoresis to separate the resulting glyoxal-RNA-ethidium adducts. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 6: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde Separation of RNAs according to size is t

11、he first stage in northern blotting and hybridization. The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels containing 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts. IMPORTANT

12、: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 7: Transfer and Fixation of Denatured RNA to Membranes This protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffer

13、s. RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers. However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 8: Northern Hyb

14、ridization This protocol describes how to carry out northern hybridization at high stringency in phosphate-SDS-buffers. Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here.IMPORTANT: Prepare a

15、ll reagents used in this protocol with DEPC-treated H2O.Protocol 9: Dot and Slot Hybridization of Purified RNA Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum

16、manifold. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 10: Mapping RNA with Nuclease S1 Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe. At the end of the reaction, nuclease S1 is used to degrade u

17、nhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by either autoradiography or Southern hybridization. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5 and 3 ends

18、of mRNAs on cloned DNA templates. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 11: Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Preparations of RNA containing an mRNA of interest are hybridized to a radiolabeled single-str

19、anded RNA probe. At the end of the reaction, a mixture of RNase A and RNase T1 is used to degrade unhybridized regions of the probe, and the surviving molecules are then separated by denaturing gel electrophoresis and visualized by autoradiography. The method can be used to quantitate RNAs, to map t

20、he positions of introns, and to identify the locations of 5 and 3 ends of mRNAs on cloned DNA templates. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Protocol 12: Analysis of RNA by Primer Extension Primer extension is used chiefly to map the 5 termini of mRNAs. A prep

21、aration of polyadenylated mRNA is first hybridized with an excess of a single-stranded oligodeoxynucleotide primer, which is complementary to the target RNA and radiolabeled at its 5 terminus. Reverse transcriptase is then used to extend the 3 end of the primer. The size of the resulting cDNA, measu

22、red by denaturing polyacrylamide gel electrophoresis, is equal to the distance between the 5 end of the priming oligonucleotide and the 5 terminus of the target mRNA.IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O.Chapter 7, Protocol 1Purification of RNA from Cells and Ti

23、ssues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction In this single-step technique, cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysate by extraction with phenol:chloroform at reduced pH. Many samples can be processed simultaneously and speedily. The

24、 yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7 g/ml starting tissue or 5-10 g/106 cells. IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H2O. CAUTIONRECIPEMATERIALSBuffers and SolutionsChloroform:isoamyl alcohol (49:1, v/v) Ethan

25、ol Formamide (Optional) Deionized formamide is used for the storage of RNA.Isopropanol Liquid nitrogen Phenol PBS Required for cells grown in suspension and monolayers only.Sodium acetate (2 M, pH 4.0) Solution D (denaturing solution) Cells and TissuesMammalian cells Mammalian tissue samples METHOD

26、1. Prepare cells or tissue samples for isolation of RNA as appropriate for the material under study. The table below describes the amounts of Solution D required for each type of sample.Amount of Solution D Required to Extract RNA from Cells and Tissues Amount of Tissueor CellsAmount ofSolution D100

27、 mg of tissue3 mlT-75 flask of cells3 ml60-mm plate of cells1 ml90-mm plate of cells2 ml2. For tissues a.Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.b.Transfer approx. 100 mg of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tis

28、sue using a pestle. The tissue can be kept frozen during pulverization by the addition of liquid nitrogen.c.Transfer the powdered tissue to a polypropylene snap-cap tube containing 3 ml of Solution D.d.Homogenize the tissue for 15-30 seconds at room temperature with a polytron homogenizer.3. For mam

29、malian cells grown in suspension a.Harvest the cells by centrifugation at 200-1900g (1000-3000 rpm in a Sorvall RT600 using the H1000 rotor) for 5-10 minutes at room temperature in a benchtop centrifuge.b.Remove the medium by aspiration and resuspend the cell pellets in 1-2 ml of sterile ice-cold PB

30、S.c.Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 2 ml of Solution D per 106 cells.d.Homogenize the cells with a polytron homogenizer for 15-30 seconds at room temperature.4. For mammalian cells grown in monolayers a.Remove the medium and rinse the cells once

31、with 5-10 ml of sterile ice-cold PBS.b.Remove PBS and lyse the cells in 2 ml of Solution D per 90-mm culture dish (1 ml per 60 mm dish).c.Transfer the cell lysates to a polypropylene snap-cap tube.d.Homogenize the lysates with a polytron homogenizer for 15-30 seconds at room temperature.5. Transfer

32、the homogenate to a fresh polypropylene tube and sequentially add 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol per milliliter of Solution D. After addition of each reagent, cap the tube and mix the contents thoroughly by inversion. 6. Vortex the homogenate vigorously for 10 seconds. Incubate the tube for 15 minutes on ice to permit comple