I have been doing the m6A dot blot for a while with no improvement, I am extracting the RNA, and I can see the dots although the three biological replicas give a different reading on the memberan while I am sure about the dilution and the similar RNA load since I confirmed the CT values by the qPCR and they were the same nearly ( Photo attached)
Any suggestions?
The procedures involve
1- Extraction of Total RNA by the Invitrogen kit
2- Dilute the RNA so that 200ng/ul is there
3- Denatutartin the RNA 95oC for 5 min then on ice till loading
4- load the RNA 5ul on the membrane ( total 1 ug/spot)
5- Air dy 15 mins
6- UV cross-linking 2 mins twice
7-Wash by PBST 5 mins
8- Block with 5% skimmed milk in PBST
9- Primary antibody 1:1000 in 5% skimmed milk in PBST over nigth in 4 C
10- Wash three times PBST 5min each
11- Secondray antibody 1:3000 n 5% skimmed milk in PBST, 2 hours in the room temperature
12- Wash three times PBST 5min each then imaging
I hope that the following points will support your study,
Clean your blotting apparatus thoroughly before performing blotting to avoid cross contamination from pervious samples.
5 microlitre sample is seems very little to blot and some quantity may stuck on the walls of blotting apparatus. It's very difficult to spread over membrane while applying vaccum. You may dilute your samples and controls up to 40-50 microlitre with nuclease free water and load it.
Dry your membrane at 37oC for 30 min to bind your RNA with membrane.
If possible, use 5% BSA instead of skimmed milk powder.
Wash your membrane 3 to 5 times with PBST on each process.
Use orbital shaker for each incubation process. Eg Blocking, primary antibody and secondary antibody.
Ensure the complete immersion of membrane with solution on each process.
Primary antibody incubation at 37oC for 1 to 1:30 hours, Instead of overnight incubation.
With reference to your image, your antibody, conjugate and substrate systems are working good.
The Substrate system not clearly mentioned.
I have done the dot blotting for viral protein and attached the paper. It will give some idea.
Thanks but doesn't 50ul of sample very large amount to load and stuck with the antibody? Also shall I increase concentration of RNA instead of 1ug?
You may increase the sample quantity, also consider the maximum detection limit of your assay. (intensity of dots).
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