Spot and slit hybridization
Spot and slit hybridization analysis of RNA enables the qualitative or semi-quantitative determination of RNA by directly spotting RNA samples onto nitrocellulose or nylon membranes without gel electrophoresis, and then hybridizing them with radiolabeled probes, which was originally used to describe spot hybridization of RNA by Kafatos et al. However, because direct spotting of RNA samples on nitrocellulose membranes produces round spots, which are not easy to compare, a device for spotting samples with a narrow slot, also known as SlotBlot, has been used.
Operation method
Spot and slit hybridization
Materials and Instruments
Deionized formamide Formaldehyde (37%) 20XSSC NaOH pre-hybridization solution Move I. Materials and equipment Caveat 1) Total cellular RNA obtained by the rapid preparation method may contaminate genomic DNA, making it difficult to accurately measure the RNA content using a spectrophotometer. It is also necessary to prevent the appearance of sample artifacts. For semi-definitive analysis, each sample should be blotted twice, one membrane hybridized with the RNA probe to be detected, and the other hybridized with another RNA probe, which should have the same expression in each sample, or you can also make a blot for a sample, but the same membrane must be hybridized twice.2) RNA samples should be diluted 8-fold at the time of blotting, so the concentration of the sample should be at least 10mg/mL.3) False positive signals can easily occur with slit hybridization. This can be avoided as much as possible by (i) rigorous characterization of the specificity of the probes by Northern blotting; (ii) the need for appropriate negative controls in all tests; and (iii) the need to prepare the probes, solutions, RNA, etc., with great care to avoid contamination. For more product details, please visit Aladdin Scientific website.
Slit Spotter Vacuum Pump Iron-sealable plastic bags or hybridization tubes Nitric acid fiber K membrane or nylon membrane Whatman3MJV1 Filter paper
1) Slit
2) Sampler
3) Vacuum pump
4) Sealable plastic bag or hybridization tube
5) Nitric acid fiber K membrane or nylon membrane
6) Whatman3MJV1 Filter Paper
7) Deionized formamide
8) Formaldehyde (37%)
9) 20XSSC
10)0.1mol/LNaOH
11) Pre-hybridization solution: 50% formamide, 5XSSC, 2XDenhardt, 0.1% SDS, 100ug/ml salmon sperm DNA.
II. Methods of operation
(-) Sample treatment
Mix l0ul of RNA10ug, (RNA can be up to 50ug) aqueous solution with the following solution:
100% formamide 20ul
Formaldehyde (37%) 7ul
20XSSC 2ul
Incubate at 60℃ for 15 min to denature, and cool the sample in ice bath.
(ii) Spotting
Wash the spotter with 0.lmol/L NaOH and rinse with DEPC water. Spread a piece of Whatman3 MM filter paper infiltrated with 20XSSC on the sampler, put a piece of nitrocellulose membrane infiltrated with 20XSSC for 30 min on top of it, put the lid on and clamp it tightly, and turn on the vacuum pump. Wash each well with 10XSSC. Add 2 times the volume of 20XSSC to each RNA sample, mix and add samples. Position the dye in the outer wells and aspirate slowly, using lml of 10XSSC twice per well. Continue suctioning for 5 min and blot the membrane.
(iii) Fixation
Remove the filter membrane, dry it at room temperature, sandwich it between two sheets of Whatman3 MM filter paper, and dry bake it at 80°C for 2 h to fix the RNA.
(D) Pre-hybridization
After drying, put the filter membrane into a plastic bag (or hybridization tube), add 20-30 ml of prehybridization solution, seal the plastic bag and prehybridize at 42°C for 3 h.
(E) Probe preparation and labeling
Refer to the previous section.
(vi) Hybridization
The labeled probes were denatured by boiling for l0 min, and then immediately placed in an ice bath for 10 min (single-stranded probes do not need to be denatured). The denatured probes were added to the prehybridization solution, and then sealed in a hybridization plastic bag and hybridized at 42℃ overnight.
(VII) Wash the membrane
After hybridization, wash the membrane with the following washing solution:
1) 2XSSC, 0.5% SDS, room temperature for 5 min.
2) 1XSSC, 0.1% SDS, 42°C for 30rnin.
0.1XSSC, 0.5% SDS, wash the membrane at 56°C for 30 min.
(viii) Detection
Radiographic autoradiography was performed on X-ray film with additional sensitizing screen, and exposed at 70°C for 7-10 days.