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Old 01-03-2020, 08:15 PM   #1
HV3450
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Location: US

Join Date: Jan 2020
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Default Normalizing RNA-seq data vs Normalizing qRT-PCR

I am working on a project to validate the results of RNA-seq differential expression analysis using qRT-PCR. I use BioRad software (CFX Maestro). I'm currently looking at genes that showed down-regulation in treated samples compared to control samples in the RNA-seq data.

When I normalize my qRT-PCR results to reference (housekeeping) genes, the results do not reflect what is seen in the RNA-seq data...they are up-regulated. However, if I choose 'Relative Quantitation' in the expression module of the software (delta-Cq without normalization) the results generally reflect what is seen in the RNA-Seq data.

What I am wondering about is whether the RNA-seq differential expression normalization using FPKM before comparisons between treatment and control reflects the same type of normalization as using a reference (housekeeping) gene in qRT-PCR.
Is this just a strange pattern I'm seeing where the normalized genes are upregulated vs when they are not normalized?
Thanks for any insight.
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fpkm, qrt-pcr, reference genes, rna-seq, validation

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