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RNA dilution

RNA dilution - Real-Time PCR and Quantitative PCR Forum

RNA dilution - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 03-09-2009, 10:29 PM
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Red face RNA dilution



Hi, I have a question here, and thank you for your attention and answer.

For the samples of total RNA with different concentration, I always dilute them to the same level for cDNA synthesis and qPCR. However, the sample with higher starting RNA concentration always gives me higher beta-actin expression level. This interferes with my data analysis. I don't know the causes and how I can avoid this?

Thanks,

Wenze
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Old 03-10-2009, 03:28 AM
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Default Re: RNA dilution

Hello, the only way to do this is use a very accurate analyzer of RNA concentration / quantity. Either UV spectrophotometry or the new microwell analyzers of DNA/RNA.
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Old 03-10-2009, 05:01 PM
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Default Re: RNA dilution

Hello Administrator,

Thank you for your quick reply. After diluting the RNA samples, I measure their concentrations again with the UV spectrophotometry to confirm that they are at the same level. However, the problem still happens. Is there any way to reduce the error in the data analysis made by the difference in beta-actin expression?

Thanks,

Wenze
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Old 03-10-2009, 05:12 PM
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Default Re: RNA dilution

Hello,
Thank you for your quick reply. After diluting the RNA samples, I measure their concentrations again with the UV spectrophotometry to confirm that they are at the same level. However, the problem still happens. Is there any way to reduce the error in the data analisis made by the difference in the beta-actin expression?

Thank you,

Wenze
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