8 replies to this topic

[gyma]

Hi, please help me clear this problem. I just extracted RNA by Trizol and did electrophoresis (non-danature). The quality should be ok according to the 28s and 18s band. and I also checked the absorbance between 200nm and 300 nm, the wave is also ok. But I noticed a weak band on top of 28s, what is that? If it is gDNA, why could I still see it after DNaseI treatment, though not very clear according to the load.


Left to Right:
Marker
RNA of cell line A (1 ul load)
RNA of cell line B (1 ul load)
DNaseI treated A (1ul RNA in 11ul total volume, loaded 3ul)
DNaseI treated B (1ul RNA in 11ul total volume, loaded 3ul)

another question:
for cDNA synthesis, should I use fresh RNA only? If I used thawed RNA from -80C, should I re-quantify the conc. by nanovue?
many thanks.

[bob1]

gDNA will usually appear as a bright band up near the wells - so long as it is not digested in some fashion.

There are other ribosomal RNA bands that can be seen besides the 28s and 18s.  It is quite likely that you are seeing one of these.

You can use stored RNA, it should be fine at -80.  I don't think you need to re-quantitate, as this will tell you nothing about the integrity of the RNA, which is the problem with storage and freeze/thaw cycles.

[gyma]

bob1, on 12 June 2011 - 03:36 PM, said:

gDNA will usually appear as a bright band up near the wells - so long as it is not digested in some fashion.

There are other ribosomal RNA bands that can be seen besides the 28s and 18s.  It is quite likely that you are seeing one of these.

You can use stored RNA, it should be fine at -80.  I don't think you need to re-quantitate, as this will tell you nothing about the integrity of the RNA, which is the problem with storage and freeze/thaw cycles.

thanks, bob1. I want to re-quantitate because I thought freezing/thawing will affect the concentration of RNA. If the conc. of RNA changed, my RT-PCR result wouldn't be believable, right?

[bob1]

Yes, but you will still have the fragments of the RNA in there which (should) give you the same reading as you had before freezing the RNA... but the RNA might be degraded, you can only see that on a gel or similar system.

[gyma]

i see, thank you very much.
Is it different from DNA? To get a reliable result in reporter assay, I have to re-quantitate my plasmid DNA every time before transfection, because the conc. decreases as time. I agree on that even if RNA is degraded, you cannot see a conc. change. But for DNA, what do you think is the reason for the conc. decrease?

[bob1]

For plasmids the concentration shouldn't decrease over time (though this might be dependent on how exactly you are quantitating - if you are using a fluorescence method, degradation will alter the apparent reading).  Degradation of the plasmid will affect how well it works in a reporter assay; nicked and linear DNA don't transfect as efficiently as supercoiled.

[gyma]

bob1, on 14 June 2011 - 04:50 PM, said:

For plasmids the concentration shouldn't decrease over time (though this might be dependent on how exactly you are quantitating - if you are using a fluorescence method, degradation will alter the apparent reading).  Degradation of the plasmid will affect how well it works in a reporter assay; nicked and linear DNA don't transfect as efficiently as supercoiled.

thank you.

[Evanescence]

Hmmm...

Yeah i agree with bob if genomic is the matter, the band should be on top near to wells, make sure you prepare this RNA in cleanest way you can do, sometimes people use slightly higher temperature while running gel(buffer) to avoid supercoil..i observed from my labmates...it did work..

but if u think it's genomic then you really need to make in special place where's less contamination around...

RNA is not like DNA/plasmid which is not quite stable..so to get nice cDNA, you should use fresh RNA...1 week is long enough..try to get is as fresh as you can...

Good luck, dont worry about the failure coz it is the way how you get something from the lesson

[gyma]

Evanescence, on 20 June 2011 - 02:40 AM, said:

Hmmm...

Yeah i agree with bob if genomic is the matter, the band should be on top near to wells, make sure you prepare this RNA in cleanest way you can do, sometimes people use slightly higher temperature while running gel(buffer) to avoid supercoil..i observed from my labmates...it did work..

but if u think it's genomic then you really need to make in special place where's less contamination around...

RNA is not like DNA/plasmid which is not quite stable..so to get nice cDNA, you should use fresh RNA...1 week is long enough..try to get is as fresh as you can...

Good luck, dont worry about the failure coz it is the way how you get something from the lesson

thank you for your comment. This RNA worked in my RT-PCR and qPCR experiments. By the way, realtime PCR is so different from RTPCR. I did pre-experiment to optimize in RTPCR before doing qPCR. However, after I got everything ok in RTPCR, I still had a lot of problems in realtime PCR. Such as primer dimer, NTC positive result etc. Lucky finally I succeeded.