frist@ccu.umanitoba.ca (02/20/91)
Has anybody come up with a way of fixing DNA or RNA to the bottoms of
microtiter plate wells? Say, for example you wanted to fix total RNA
samples from a large number of tissue samples (eg. a timecourse
experiment), and then hybridize with a biotinylated probe. Next add
streptavidin-conjugated alkaline phosphatase, and substrate, and read the
result in your microtiter plate reader. Essentially we're talking about
an ELISA for DNA.
---------| |-----------
| %%%%% | streptavidin-conjugated enzyme
\ ---- / biotinylated antisense RNA probe
\ ~~~~ / unlabeled total RNA (fixed to plate)
------
This is such an obvious idea that I would think there must be a kit
somewhere for it, but I don't recall having seen it in the literature.
===============================================================================
Brian Fristensky | What can literature do against the pitiless
Department of Plant Science | onslaught of naked violence? Let us not for-
University of Manitoba | get that violence does not and cannot flourish
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frist@ccu.umanitoba.ca | LYING... Lies can stand up against much in
Office phone: 204-474-6085 | world, but not against art.
FAX: 204-275-5128 | Alexander Solzhenitsyn, NOBEL LECTURE
===============================================================================kim@m44.unm.edu (02/21/91)
The kind of assay you described seems to be functionally similar to a dot blot/slot blot, except that slots cannot be read by a microplate reader. A really indirect sandwich that may work would be to allow avidin to adhere to the bottoms of the wells, and then use biotinylated total RNA (Maybe photobiotinylated). Or, to be even more indirect, fix avidin to the plate, then put biotinylated oligo-dT into the well for the second layer of the sandwich. Wash the well, then apply your total RNA. Poly(A) RNA will then hybridize with the oligo(dT). Do another wash, not disrupting the hybrids, and then add your probe. Daniel Kim
BIOLEV@UNIPAD.UNIPD.IT (Max) (02/21/91)
>Has anybody come up with a way of fixing DNA or RNA to the bottoms of >microtiter plate wells? Say, for example you wanted to fix total RNA >samples from a large number of tissue samples (eg. a timecourse >experiment), and then hybridize with a biotinylated probe. Next add >streptavidin-conjugated alkaline phosphatase, and substrate, and read the >result in your microtiter plate reader. Essentially we're talking about >an ELISA for DNA. >---------| |----------- > | %%%%% | streptavidin-conjugated enzyme > \ ---- / biotinylated antisense RNA probe > \ ^^^^ / unlabeled total RNA (fixed to plate) > ------ >This is such an obvious idea that I would think there must be a kit >somewhere for it, but I don't recall having seen it in the literature. Yes, well I actually am using such a type of assay to detect Benzo(a)pyrene DNA adducts using an ELISA assay with a policlonal antibody raised against the BP-DNA adducts. The DNA samples are fixed to the bottom of the microtiter plate by first heat-denaturing them and then placing the solution in the wells and allowing the wells to dry out overnight at 37 C. The DNA then binds to the wells fairly well (say 100 ng DNA/well). Then incubate the first antibody with the DNA containing wells and after this incubate a second antirabbit-alkaline phosphatase conjugate and then use either a fluorochrome (methylumbelliferyl) or PNPP for visible EIA readers (405 nm). This is just a quick run through the method. If any further details are required......Glad to be of help!!!!!