non-radioactive

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i am doing an experiment that involves in situ...

past experiments used radioactivity to locate mRNA...

mine doesnt.. why is that better? what's wrong with using radioactive stuff?

please help!

thanks

 

[bgreet]

i am doing an experiment that involves in situ...

past experiments used radioactivity to locate mRNA...

mine doesnt.. why is that better? what's wrong with using radioactive stuff?

please help!

thanks

Umm what are we comparing it to? It would help knowing what the new marker is.

 

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labeling mRNA using radioactivity vs. nonradioactive labeling...

why would nonradioactive be better?
what are the effects of radioactivity?

 

[Pinkertinkle]

You gotta be more careful with the radioactive stuff?

 

[bgreet]

Umm possible base changes? I really dont know, and what nonradioactive marker system are you using? Non radioactive isotopes?

 

[principessa]

Any time you can avoid working with radioactive materials, you want to. They're more dangerous to you, and you have to take all sorts of precautions in order to work with them - wipe tests, disposal of waste in a special container, etc.

 

[Medikit]

Why do you want to work with radiation if you don't have to?

What are you using instead?

 

[Treg]

labeling mRNA using radioactivity vs. nonradioactive labeling...

why would nonradioactive be better?
what are the effects of radioactivity?

Ok, if you are doing in situ hybridization, you are looking for location of your RNA in the cell, right? If you use non-radioactive, i.e. a fluorescent tag, you can detect your RNA and relate it to organelle location, by using a complementary fluorophore like mitotracker or a cytoskeletal marker and doing confocal microscopy. Radioactive probes are usually used to identify whether certain RNAs are present in terms of molecular weight in cell lysates, i.e. RNAse protection assays or Northern blots. In this case, which is not in situ, you can make a radioactive probe and detect the RNA you are looking for on a blot transferred from a gel you run.

Does that answer your question?

🙂Treg

 

[OrthoFixation]

what are the effects of radioactivity?

I think it actually cooks your insides. It is measured in Roentogens or millirems in small doses. I worked in a nuclear power plant for several years. The less, the better. An extreme example is the pilots who flew over Chernobel dumping sand on the reactor lived on average about 8 hours due to the high doses received 😱

 

[vcatz]

In situ is commonly used to determine where in a tissue of interest a particular mRNA is expressed. The older, more common techniques utilize 35S, 32P, or 33P labeled oligos or riboprobes - these are the isotopic methods. They usually work quite well in my experience. There are also kits available that utilize HRP/chemiluminescense instead of radioactivity. They usually invovle labeling a nucleic acid probe with dig, hybridizing it to the tissue, and then using a HRP conjugated anti-dig antibody to visualize the hybridized probe. Some of these methods work pretty well but in my experience they do not work as well as the older methods. That being said there are many labs who generate beautiful in situs using the non-isotopic methods - C. Thisse comes to mind. The amount of radioactivity you use in the older protocols is nominal (especially compared to a nuclear accident) and does not present a risk to the experimenter. As the OP said - you just have to be more careful with the isotopes.

 

[EvoDevo]

Also don't forget that in cases where the gene expression being assayed is relatively weak, use of the radioactive protocols is actually preferred. The relative molar amounts of mRNA necessary to generate a signal in HRP/chemiluminescense is >> radiolabeling.

 

[vcatz]

I totally agree - that is why I prefer 33p riboprobes over any of the other techniques

 

[uproarhz]

I think it also depends on what mRNA you are looking for. For example if you are working with anything that is going to be affected by DNA damage, your results may be confounded because radioactive isotopes, even at really low experimental doses that don't pose a threat to the experimenter, actually generate a good number of DNA double strand breaks in the cells being labeled. Well, I mean not a ton, but enough to initiate a DNA damage response. Just something to keep in mind. But I doubt this is your case.

 

[synapse]

ahh u guys... its not any of that.. i just told my pi..

"its cuz...the mrna will degrade faster if u use a radioactive label... also, if u use a radioactive label.. u have to be more careful"

but yeah, the answer is morphology.

:*(

 

[jlee9531]

ahh u guys... its not any of that.. i just told my pi..

"its cuz...the mrna will degrade faster if u use a radioactive label... also, if u use a radioactive label.. u have to be more careful"

but yeah, the answer is morphology.

:*(

the simplest answers always the best i guess.