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Help with RT-PCR, late curves

Help with RT-PCR, late curves - Real-Time PCR and Quantitative PCR Forum

Help with RT-PCR, late curves - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 09-12-2012, 08:29 PM
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Unhappy Help with RT-PCR, late curves



Hi All,
I am new to the forum and RT-PCR. In my experiment the HK gene amplifies at 30 cycles but my target gene starts at 40-45, which I think is quite late.
What should I do? I have tried different concentrations of cDNA and repeated the RT on the samples.
The target gene is expressed low in the tissue, according to the litreature.
Is the data obtained after 30 cycles still usefull?
Can anyone suggest another Housekeeping gene which starts amplifying, lets say after 35-40cycles?
Thanks in advance,
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Old 09-17-2012, 06:13 PM
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Default Re: Help with RT-PCR, late curves

is it human HK gene? which one?, we are using RP assay for detection of Human RNase P, we usually got around middle of 30 of Ct value from original human nasal swap specimens, is it what you are looking for?
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Old 09-18-2012, 12:53 AM
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Default Re: Help with RT-PCR, late curves

Importantly have you run a gel of the said 40-45 cycles (gel to show correct size amplification) or the amplification is based on peak on the RT system?
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Old 09-18-2012, 04:11 AM
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Default Re: Help with RT-PCR, late curves

Hello,what's the reagents you're using?
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