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multiple bands in my RNA sample?


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#1 hpgrds

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Posted 14 April 2009 - 07:29 PM

Hi

I am new on RNA stuff. I isolated RNA from my bacteria but I got so weird results. I got 4 or 5 bands in 1% formadehyde denature gel. Or I got at least 7 bands in 2% agarose gel. I don't know what it means. Does this suggest that my RNA are degraded. But does degraded RNA look like smear instead of clear band? I attach the picts in this post. the first pict shows RNA in formadehyde denature gel, and I used 1 Kb DNA ladder. The first band is close to 3Kb, and the second band is close to 2Kb. In the second pict, the last lane shows RNA run in 2% agarose gel, and the ladder is 100 bp DNA ladder. The first band might be 1.2 kb, the second band might be 700bp. Since this is agarose gel and DNA marker, I don't think the size is accurate, but I got more bands in agarose gel compared with denature gel. ( I did heat up my RNA sample with loading buffer @ 65C for 15 mins). Does someone have any idea what it is happening? Thanks so much!

hpgrds

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  • pcr_genomic_DNA_and_RNA__2nd_digested___3rd_preticipation__resize.jpg
  • totall_RNA_4_8_resize.jpg


#2 Carlton H

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Posted 17 April 2009 - 12:19 PM

If you're new to RNA stuff, it's distinctly possible that you contaminated one of your RNA samples, and yes, partially degraded RNA could form a big smeary band like the one you see. Know that as an evolutionary defense against retroviruses, you practically ooze RNAse, so make sure you wear gloves, either wash & sterilize your equipment or use designated "RNAse-free" equipment that's been handled such as not to get contaminated with RNAse, don't breath on you samples, don't talk in their direction, etc, etc, etc.

Aside from that, go back over your meticulously recorded lab notes and see if there's anything else you did differently between the two gels.

-Carlton
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#3 mastermi

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Posted 17 April 2009 - 11:14 PM

For me this looks more like DNA.

I isolated RNA from different bacteria, and they always look quite similar: You get two distinct bands for ribosomal RNAs and one lower smeary band which is the tRNAs.
If your RNA is degraded you will see smear and not distinct bands.
Since you can't distinguish DNA from RNA on gels or photometrically, I would guess that you have DNA in your sample. Although I don' know why this DNA shows several bands.

How are you isolating your RNA? If you use phenol, did you make sure that it is acid phenol?




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