Northern blot is a method to detect gene expression by detecting the expression level of RNA. Firstly, different RNA molecules are distinguished according to their molecular weight by electrophoresis, and then the target fragments are detected by hybridization with probes complementary to specific genes. Northern blotting is actually named after Southern blot, a hybridization technique invented earlier than it. Southern blot is mainly used to analyze DNA.
Northern blot can be used to detect the expression patterns of specific genes in different tissues, organs, different development stages of organisms, stress environment or pathological conditions, and also can be used to detect whether the target gene has variable splicing products or repetitive sequences.
Under certain conditions, two nucleic acid single strands with certain homology can form double strands according to the principle of complementary bases. This hybridization process is highly specific. Under the condition of denaturation, RNA samples were examined by agarose gel electrophoresis, then transferred to the membrane and hybridized with probes to detect the expression level of RNA.
1.Total RNA extraction;
2.Preparation of modified adhesive:Take 0.2g of agarose, add 12.4ml of DEPC water, and heat to melt,in the pre insulation state, add 5 * formaldehyde gel electrophoresis buffer 4.0mL, 37% formaldehyde 3.6ml, mix and make glue. After gelation, 1 * formaldehyde gel electrophoresis buffer was used to pre electrophoresis 5min.
3.Sample preparation:The total RNA was about 20-30 μg, added 5*formaldehyde gel electrophoresis buffer solution 4.0 μl, 37% formaldehyde 3.6 μl, formamide 10 μl, incubated at 65 degrees 15min, ice bath 5min. EB（1μ g/μl） 1μl and loading buffer 2μl were added.
4.Electrophoresis:Sample loading, 50 V electrophoresis (electrophoresis time about 2 hours),after electrophoresis, the gel block was placed under the ultraviolet light to observe the integrity of RNA and record the position of 18S and 28s bands(distance from sampling hole).
5.RNA was transferred from denatured gel to nitrocellulose or nylon membrane.
6.The above RNA transfer lasted about 15 hours.In the process of film transfer, when the paper towel is wet, it should be replaced with a new one.
7.After the transfer, remove the towel and 3MM filter paper above the gel.The membrane was immersed in 6*SSC for 5 min to remove the residual gel on the membrane.
8.The gel was placed under the ultraviolet lamp to observe whether there was any residual RNA on the gel block.
9.The film was placed at 80 ℃ and dried in vacuum for 1-2 hr.The dried film was sealed with plastic bag and stored at 4℃.
10.Probe labeling.
11.Pre hybridization:The reverse side of the membrane was tightly attached to the hybridizing bottle, 5ml of pre hybridizing solution was added, and pre hybridized at 42 ℃ for 3 hr.
12.Hybridization:The denatured probe(denaturation at 95-100 ℃ for 5 min and ice bath for 5 min) was added into the pre hybridized solution and hybridized at 42 ℃ for 16 hr.
13.Washing membrane.
14.Tablet pressing.
Related reading： southern blot protocol Introduction To Southern Blotting