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RNA dot blot


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6 replies to this topic

#1 rephi

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Posted 14 October 2009 - 12:16 AM

Hi! I want to detect Bacterial mRNA using dot blots. Can anyone give me hints, where to find good protocols for labeling probe DNA/RNA, and/or for RNA dot blots in gerneral? I was only able to find stuff about the protein dot blots, besides the wikipedia article :( I would be very glad about any suggestion!

regards
rephi

#2 phage434

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Posted 14 October 2009 - 04:06 AM

Step 1 is to determine your detection method. Is it radioactive or fluorescent? If radioactive, you can use normal primers and kinase with 32P ATP, hybridize, and detect with film or screen. This approach is definitely out of style in most labs in an effort to get rid of radioactivity. If fluorescent, you can do things in several ways. Simplest is to order your probe with a 5' fluorescent label, but this is expensive. More commonly the probe is ordered with a biotin or digoxigenin (DIG) label, hybridized, and detected with a fluorescently labeled antibody to biotin or dig. More complex approaches hybridize, and detect with antibody linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. The enzymes are then detected with luminescent systems such as ECL. I would start with DIG labeled oligos and detect with horseradish peroxidase linked antibody and luminescent detection. If you have a chilled CCD imager, then detection is fast and efficient. If not, then film with still work. The place to start reading about this is probably some vendor. Roche and Thermo/Pierce come to mind as two of the major players in this area.

#3 rephi

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Posted 29 October 2009 - 02:46 AM

thank you for your answer! as i first step i will try to use dna for blotting instead of rna, just to try out the technique...
I think about using biotinylated primers for labeling my dna. In my mind and according to what I have read sofar the experiment would work something like this:

1. Crosslink Traget DNA to Ny+ membranes (total bacterial DNA)
2. PCR with biotin labeled primers of specific squence
3. Hybridize PCR product from (2) with DNA on membrane from (1)
4. Detection


Is this correct? Do you have any suggestions? I am trying to find protocols for the different steps for quite some time now and I would be glad for hints.

regards, rephi

#4 phage434

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Posted 29 October 2009 - 04:03 AM

You only need biotin on one of your two pcr primers. You may not need to do pcr at all, and could just use a relatively long oligo as the probe. You should debug this from the probe detection back to the hybridization. First, crosslink your biotinylated pcr product to the membrane in serial 10x dilutions, and get your detection method working on this simple system. Only then should you worry about hybridization and all of its difficulties and variables. Run lots of controls. I would routinely run a dot blot of the serial probe dilutions -- this tells you immediately whether the problem is a detection or a hybridization problem.

#5 rephi

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Posted 29 October 2009 - 05:15 AM

thanks again! i wasn't very clear with my explanation, I only ordered the forward primer with biotin label.
i am not very experienced with blotting techniques, maybe therefore it is difficult for me to explain what I actually need. Du You have any reading suggestions for me, to get better insights in thsi topic? I am relatively alone in my lab, because nobody has experiece with this either. I also came across this Fermenats Kit: http://www.fermentas...otinchromog.htm

#6 phage434

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Posted 29 October 2009 - 05:21 PM

See, for example, the DIG manual for filter hybridization by Roche, here:
https://www.roche-ap...MAN/dig_toc.htm

You can replace DIG with Biotin with much the same effect, or check out the manuals at other manufacturers.

#7 rephi

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Posted 02 November 2009 - 12:26 AM

thank you again!




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