Hi!

These spots are appeared from degrdation of radioactive label. In our lab we obtained these spots after insufficient purification of radioactive probe. The probe become contaminated with radioactive nucleotides that are readily hydrolysed upon heating and 32P is released are non specifically binds to membrane. To get rid off spost:

1.check your probe for the presence of degradation products. e.g. PAAG electrophoresis, if you'll see a smear - change the probe.

2. make additional purificaton of your labeled probe to remove contaminated radioactive label

This happened in the past in my lab and it was due to the humidity in the cassette. After we dry them well these spots disappeared. However it is well possible that also happens what is explained above. We now use only qReal-Time pCR.

check the hybridisation membrane. some companioes have introduces changes and e.g. certain hybond membranes are not suitable for use with 32-P anymore although previously they were. Applichem (Darmstadt, Germany) manufactures a very nicely suitable positively charged nylon membrane with the help of which I got rid of such background. You can also try to use the good old Church-buffer instead of some commercial kits (sometimes helps).

Make also sure that the oven and any glassware you are using is pre-heated before starting even pre-hybridisation, this can also help to prevent background.

Thank you all for the helpful replies. I actually do not heat the probe prior to hybridization but this is a probe that I make by IVT so maybe it is a good point to gel purify instead of the RNA clean up kit I've been using. I do however remove the unincorporated ATP using a spin column.

I've gotten spots like these under two conditions: (1) prehybridizing for too short a period of time (even with the rapid hyb buffers) and (2) bruising the membrane by prehybridizing/hybridizing too roughly. Pre-hybridize longer with gentler agitation; then check for background with a geiger counter before exposing. I don't think gel purifying your oligo is necessary.

It looks like precipitates of the probe that then sticks and aggregates. So take the advice suggested to remove any precipitation, like heating. It might be wise to not just spin down but actually filter the probe solution in a 0.22 um spin filter before use and keep temps constant, as was also suggested. I do also highly recommend gel-purification of the radiolabeled probe for all applications to get very specific and reproducible results.

Really it’s quite dirty this membrane.

When in the past we used the technique of northern-blot in our lab, sometimes appeared this inconvenience.

This was remedied when we worked with all the solutions filtered through millipore 0.45 to remove any insoluble powder that sometimes contaminates solutions.

Eliminate all gloves with powder (use only powder-free gloves in your lab).

Filter all solutions in 0.45u millipore.

Filter your radioactive probe through P10 columns to remove unincorporated isotope.

This should improve your results.

Good luck

PS: Try to change your expression analysis to qRT-PCR, microarrays and/or RNAseq.

Try use qReal-time PCR? Less tedious.

there is still some important info that can be learned from northern blots that you can't get with qPCR. like transcript size or number of species and their relative abundance. But if these are not important to you and you only care about expression levels, then qPCR is def the way to go.

hi again, a very quick and easy (and efficient) clean-up method for the probe is to run it over a self made sephadex spin column. depending on the size of the oligo or dsDNA probe one should obviously use G-25 or G-50. Pre-swell sephadex in TEN buffer by autoclaving. Use old (and thoroughly claened!) mini-prep columns or punch a hole into a 0,7 ml PCR tube, place a piece of Whatman 3M paper over the tiny hole (e.g. 2x2 mm piece), place the tube into a regular 1,5 ml eppendorf, fill the 0,7 ml tube with sephadex slurry, spin @ 1500-2000 g for 30 sec, discard flowthrough and repeat this step until the 0,7 ml tube is filled. Such columns may even fall dry, in which case they should be pre-wet with TEN buffer prior to use - make sure to always discard the TEN-flowthrough. As soon as no flowthrough passes the column anymore, pipette your probe onto the column, right into the centre and spin 1500-2000 g for 4 min. All of this may be done at room temperature (20-25 °C). Now the flowthough can directly be boiled and added to the hybridisation mix.

I am also doing qPCR but unfortunately for what I'm doing I need to do a northern as well. My probe is cleaned from unincorporated p32 using a spin column. I am using a commercial solution for the pre-hyb and the hyb so I assumed I do not need to filter it. I am using Roche positively charged membrane (expiration date still good). Does anyone think the membrane can be a problem?