Dot hybridization and narrow line hybridization of purified RNA
The dot and narrow-line hybridization technique (Kafatos et al. 1979) is used to immobilize several nucleic acid samples on the same solid-phase support (usually a charged nylon membrane) and then hybridize the immobilized samples with a suitable probe, from which the concentration of the target sequence is determined. The amount of target sequence in the sample to be tested is determined by estimating the intensity of the signal emitted from the sample spot and comparing it with the signal intensity of a standard of known concentration. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Dot hybridization and narrow-line hybridization of purified RNAs
Principle
The dot and narrow line hybridization technique (Kafatos et al. 1979) is used to immobilize several nucleic acid samples on the same solid phase support (usually a charged nylon membrane) and then hybridize the immobilized samples with a suitable probe and determine the concentration of the target sequence. The amount of target sequence in the sample to be tested is determined by estimating the intensity of the signal emitted from the sample spot and comparing it with the signal intensity of a standard of known concentration.
Materials and Instruments
RNA assay samples, standards and negative controls Probe labeling and denaturation Move I. Materials For more product details, please visit Aladdin Scientific website.
NaOH prehybridization solution RNA denaturing solution SSC
Blotting device Stranding device Positively charged nylon membrane Thick blotting paper Water bath
1. Buffers and solutions
NaOH ( 10 mol/L)
Pre-hybridization solution
RNA denaturing solution
0.1X SSC (containing 0.1% SDS, m/V)
0.1X SSC (with 1% SDS, m/V)
0.5X SSC (with 0.1% SDS, m/V)
1X SSC (with 0.1% SDS, m/V)
2X SSC
20X SSC
2. Nucleic acids and oligonucleotides
RNA Assay Samples, Standards and Negative Controls
3. Probes
Probe labeling and denaturation
4. Specialized equipment
Blotting device
Stratalinker (e.g. Stratalinker, Stratagene; GS Gene Linker, Bio-rad) or vacuum oven, microwave oven
Positively charged nylon membrane
Thick transparency paper (e.g. Whatman 3 MM, Schleicher&Schuell GB004, or Sigma QuickDraw)
Preheat the water bath to 68°C
II. Methods
Mounting the blotting device
1. Cut a piece of positively charged nylon film of appropriate size. Mark the direction with a pencil. Briefly wet the membrane with water and soak it in 20X SSC for 1 h at room temperature.
2. While the membrane is soaking, carefully wash the blotting device with 0.1 mol/L NaOH and then with sterile water.
3. Wet two pieces of thick filter paper with 20X SSC and place them on top of the vacuum.
4. Insert the sample tank into the upper part of the device, place the wet nylon membrane at the bottom of the sample tank filling hole, and roll a pipette over the surface of the membrane to remove air bubbles in the sandwich between the membrane and the device.
5. Tighten the clamp and connect the vacuum hose.
6. Add 10X SSC until the liquid level is no longer above the nylon membrane, turn off the vacuum, and fill with 10X SSC.
RNA Sample Preparation
7. Mix each RNA sample (dissolved in 10 μl of water) separately with 30 μl of RNA Denaturing Solution.
8. Incubate at 65°C for 5 min, then cool on ice.
9. Add an equal volume of 20X SSC to each sample.
10. Gently aspirate 10X SSC into the blotting device until the nylon membrane is submerged.
Blotting of RNA Samples and Immobilization of RNA on Membranes
11. Gently add all samples to the slit line and gently blot the membrane. After all samples have been spread onto the membrane, wash each narrow line twice with 1 ml of 10XSSC.
12. When the second wash is complete, continue to gently blot the membrane.
13. Remove the membrane and immobilize the RNA on the membrane by UV crosslinking, baking or microwave irradiation.
Hybridization of immobilized RNA and membrane washing
14. Place the membrane in a baking tray or hybridization oven, add 10-20 ml of prehybridization solution and incubate at 68℃ for 2 hours.
15. Add the denatured radiolabeled probe directly into the prehybridization solution. Continue to incubate for 12-16 h at the appropriate temperature.
16. After hybridization, remove the membrane from the plastic bag and transfer it as quickly as possible to a plastic container containing 100-200 mJ of 1X SSC (0.1% SDS) at room temperature. Cover the plastic container tightly and gently shake it on a shaker for 10 min.
17. Transfer the membrane to another plastic bag containing 100-200 ml of 0.5X SSC (0.1% SDS) preheated to 68°C and shake gently for 10 min at this temperature.
18. Repeat step 17 twice for a total of three washes.
19. Blot the film with filter paper and radiographically develop the film at -70°C for 24-48 h (Kodak XAR-5 or equivalent). Tungstate-type sensitizing screens work better than the older rare-earth types. Of course, phosphor imagers can also be used for imaging.