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RNA gel electrophoresis

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  • 20-03-2010 5:20pm
    #1
    Ronanc1
    Registered Users Posts: 397 ✭✭


    heya guys just wondering could any of you help me out heres a picture of the end result of RNA gel electrophoresis, total rna was extracted from hep2 cells and electrophoresised just wondering if any of you could help me say some more about my lane, lane 3
    untitled.JPG

    ive said possible rna'ase degradation little smearing definite band 28s band? not sure what else to say
    Thanks guys


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    #2
    Tree
    Moderators, Science, Health & Environment Moderators Posts: 4,678 Mod ✭✭✭✭Tree


    Dunno, RNA isnt my thing. I suppose the absecne of product in some lanes is noteworthy. Do you mean the third lane including your MW marker?


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    #3
    Ronanc1
    Registered Users Posts: 397 ✭✭Ronanc1


    Sorry should have made that clear each lane is done by one person mine was lane 3 some are empty because thats the way the numbers in the class worked out


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    #4
    adamski8
    Closed Accounts Posts: 4,503 ✭✭✭adamski8


    well looks like your product is off the scale of your ladder. whats the ladder size and your intended product size. you alos didnt mention what your primers were for?


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    #5
    darjeeling
    Registered Users Posts: 962 ✭✭✭darjeeling


    You said it's total RNA from HEp-2 cells (i.e. a human-derived cell line)?

    In that case, if the RNA were not degraded, I'd expect to see a bright 28S rRNA band, a less bright 18S rRNA band that has migrated further, and a fainter smear of mRNA around 0.5Kb-6Kb in size that extends either side of the rRNA bands.

    If the RNA is degraded (generally by RNAses), then you can lose the 28S and 18S rRNA bands, and end up with a faint, low molecular weight smear.

    f00286.gif
    This photo from Ambion shows two RNA preps alongside a DNA size ladder containing fragments of 0.5Kb to 9Kb in size.

    You'd need to know the sizes of the fragments in the DNA marker ladder in your gel to be able to interpret what's going on in the RNA lanes.


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    #6
    Kevster
    Closed Accounts Posts: 2,980 ✭✭✭Kevster


    My comments are:

    There's very little amplification there, but there IS some. Maybe the starting concemtration of RNA was too low. That top band (which is present in the other samples) is present in yours, but it's faint. The smear at the bottom is non-specific binding and/or a primer-dimer (I think... ...?).

    You seem to have run the gel for too long too.

    Kevin


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    #7
    darjeeling
    Registered Users Posts: 962 ✭✭✭darjeeling


    There seems to be a bit of confusion in this thread. A couple of posts mention primers and amplification products. The original post, though, tells us that the attached photo shows an agarose gel loaded with total RNA extracted from cells, with no PCR amplification step. There should therefore be no PCR product and no primers or primer-dimer artefacts. Am I right?


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    #8
    adamski8
    Closed Accounts Posts: 4,503 ✭✭✭adamski8


    ah now i see, I've got DNA gels on the brain


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    #9
    Kevster
    Closed Accounts Posts: 2,980 ✭✭✭Kevster


    darjeeling wrote: »
    There seems to be a bit of confusion in this thread. A couple of posts mention primers and amplification products. The original post, though, tells us that the attached photo shows an agarose gel loaded with total RNA extracted from cells, with no PCR amplification step. There should therefore be no PCR product and no primers or primer-dimer artefacts. Am I right?

    Would there be enough RNA in a cell population to give such strong bands? The OP didn't mention any amplification step, but that is the general step to perform after a nucleic acid extraction before electrophoresis. RNA itself is in much less quantity than DNA in cells, and I really don't think there'd be enough to give such bright bands.

    Kevin


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    #10
    darjeeling
    Registered Users Posts: 962 ✭✭✭darjeeling


    Kevster wrote: »
    Would there be enough RNA in a cell population to give such strong bands? The OP didn't mention any amplification step, but that is the general step to perform after a nucleic acid extraction before electrophoresis. RNA itself is in much less quantity than DNA in cells, and I really don't think there'd be enough to give such bright bands.

    Kevin

    Total RNA ought to be visible on an EtBr agarose gel, and the photo I previously borrowed from a commercial company (link)I posted was just such a gel. The two ribosomal RNA bands should be very bright, as rRNA accounts for around 80% of cellular RNA. I can't see two bands in any lane of the OP's gel, though, and I'm not sure what the single bright band is at the top of some lanes. One of the rRNAs? Genomic DNA contamination? Without the sizing information, I couldn't say.

    Having said all that, I'll now admit to not having run an RNA gel myself since an undergrad practical years ago, and I'm not sure if anyone nowadays uses gel-based quality assessment and quantitation of RNA as opposed to UV spectrophotometry or fluorophore labelling and detection.


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    #11
    Ronanc1
    Registered Users Posts: 397 ✭✭Ronanc1


    oops sorry guys i hadn't realized that this thread was still ongoing i seem to have stopped getting emails about responses:confused: thanks for the interesting reading tho:D ive already submitted my writeup so ill just wait and see what happens

    thanks


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    #12
    cgc5483
    Registered Users Posts: 638 ✭✭✭cgc5483


    Ronanc1 wrote: »
    heya guys just wondering could any of you help me out heres a picture of the end result of RNA gel electrophoresis, total rna was extracted from hep2 cells and electrophoresised just wondering if any of you could help me say some more about my lane, lane 3
    untitled.JPG

    You failed (to load as much nucleic acid on the gel as the first 2 people) ;)

    ive said possible rna'ase degradation little smearing definite band 28s band? not sure what else to say
    Thanks guys

    Probably correct although hard to say. Was it a denaturing agarose gel (did you use formaldehyde in the gel)? If not its difficult to interpret.
    Kevster wrote: »
    Would there be enough RNA in a cell population to give such strong bands? The OP didn't mention any amplification step, but that is the general step to perform after a nucleic acid extraction before electrophoresis. RNA itself is in much less quantity than DNA in cells, and I really don't think there'd be enough to give such bright bands.

    Kevin

    V easy to detect the 28S and 18S. On a denaturing agarose gel from about 10 ug of total RNA the 28 and 18S would be glowing. Done it many times when using Northern blots to quantify mRNA in the days before real time PCR (well in truth before labs could afford real time machines). Was always a good indicator as to whether or not i was going to waste the next couple of days.
    darjeeling wrote: »
    Total RNA ought to be visible on an EtBr agarose gel, and the photo I previously borrowed from a commercial company (link)I posted was just such a gel. The two ribosomal RNA bands should be very bright, as rRNA accounts for around 80% of cellular RNA. I can't see two bands in any lane of the OP's gel, though, and I'm not sure what the single bright band is at the top of some lanes. One of the rRNAs? Genomic DNA contamination? Without the sizing information, I couldn't say.

    Yep thats how it should look. Looks like some genomic DNA at the top alright which is usual depending on how the total RNA was extracted.


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    #13
    darjeeling
    Registered Users Posts: 962 ✭✭✭darjeeling


    cgc5483 wrote: »
    Was it a denaturing agarose gel (did you use formaldehyde in the gel)? If not its difficult to interpret.

    Good point.


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    #14
    ilovelamp81
    Closed Accounts Posts: 13 ✭ilovelamp81


    Probably a bit late on this one but Ive done a load of Northern blots and to just check the quality of the RNA its perfectly ok to run an aliquot on a DNA gel..so no need for the toxic formaldehyde/formamide etc. This just makes life easier to visualise the gel. Most people would spec their RNA on a nanodrop/spec but it doesnt indicate the quality of the RNA as well as running it on a gel.

    Hope this two cents helps:D


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