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DNA Contamination in RNA?


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#1 vwracer06

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Posted 10 January 2003 - 10:58 AM

If I do PCR on my RNA sample and get a band at a different size than I would from doing RT-PCR what does that mean?

I just want to know if the band that I got from RT-PCR is from RNA or from DNA contamination.

#2 hula

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Posted 12 January 2003 - 12:57 PM

Hello,

The extra band may mean many things. It may be just nonspecific product due to nonspecific priming. It may be DNA contamination or unknown alternative splicing forms of your mRNA. How to tell which is which? One thing you can do is to check the gene structure of your gene (go the ensembl or UCSC) to find what is the expected product size of genomic DNA amplified with your RT-PCR primers. The size should be your RT-PCR product size plus intron(s) over which your primers spanning. If the extra band is DNA contamination, the size should match the one you get from genome sequence. You can also check if there are any known alternative splicing forms. If not and the band is reproducible, do a sequencing of your band to see if it is a real isoform.

Cheers,

Hula




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