I am working on a gene expression experiment using SYBR green RT-PCR. I see perfect single peak melting curves for reference gene in my control samples but curves are erratic for treatment sample groups.I tried multiple reference gene primers,the results are the same. I am planning to spike equal amount of RNA to my extracted RNA samples for the expression study. Any one has faced similar problem? have any experience with spiking RNA for real-time RT-PCR? need your comments and suggestion. Thanks.
Are you diluting your cDNA? Undiluted cDNA might cause that especially if the curves look fine for your control samples.
Also, are you isolating the RNA in the same conditions with the same reagents?
Yes, RNA isolation conditions are same for all samples and I am directly using RNA (200ng/reaction) for quantification.
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