I persistently get a band in the negative control when trying to isolate RNA from cell samples and am wondering if it's contamination in the experiment, genomic contamination, or just the amplification of cDNA by the polymerase used in the qPCR. The negative control is obtained by using water instead of reverse transcriptase during cDNA synthesis. Interestingly, when I use cell samples, I am successful in not having a negative control in some of my samples.
Tissues: kidney cortex
Cells: collecting duct cells
RNA isolation method:
Cells: Syringe/RNEasy kit
Tissues: Motor homogenizer/Chloroform/Trizol
cDNA synthesis method: First Strand SuperScript 3
qPCR: PrimePCR from Biorad
Image 1: qPCR product gel for cell samples (last band on the right = negative control)
Image 2: qPCR product gel for tissue samples (lanes 3,8,12)
The expression of the cell negative control is, however, much lower in the qPCR than for the other samples:
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