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I HATE real-time quantitative RT-PCR. For real. Anyways, here is my question:
I get my RNA, measure concentration in triplicate by OD (spec) and then dilute out accordingly. Then I perform RT and run my samples on icycler (biorad) with SYBR Green. My control is 18s RNA. Now, since I normalize my RNA and assume that RT should not change anything, how come I'm seeing huge (2-7 fold) increase in 18sRNA levels? Shouldn't it not change, given I standardize RNA concentration prior to RT and most (95%+) of the RNA measured by spec is ribosomal RNA? What the hell am I missing here? Anybody else ran into this problem?
I get my RNA, measure concentration in triplicate by OD (spec) and then dilute out accordingly. Then I perform RT and run my samples on icycler (biorad) with SYBR Green. My control is 18s RNA. Now, since I normalize my RNA and assume that RT should not change anything, how come I'm seeing huge (2-7 fold) increase in 18sRNA levels? Shouldn't it not change, given I standardize RNA concentration prior to RT and most (95%+) of the RNA measured by spec is ribosomal RNA? What the hell am I missing here? Anybody else ran into this problem?
