What are the most common artifacts of cDNA synthesis with SMARTer kits?
What are the most common artifacts of cDNA synthesis with SMARTer kits?
Elevated baseline in the Bioanalyzer trace. This is commonly due to the presence of SPRI beads in the cDNA preparation. Although SPRI beads themselves do not fluoresce (nor will they bind the dye included in the Agilent High Sensitivity DNA Kit), any DNA remaining on the bead will bind dye and fluoresce. To prevent bead-carryover: Leave the sample on the magnetic stand for an additional five minutes to attract all beads out of the solution and onto the walls of the tube. Remove the solution very slowly, using a long pipette tip. The smaller width of the tip allows for more distance between the beads and the tip, reducing the likelihood of disturbing the beads back into solution. The electropherogram exhibits a broader peak, abnormally high yield, and/or shows multiple peaks. This usually indicates contamination. A common source of contamination is the SPRI beads, which may adsorb air pollutants (e.g., pollen). To prevent contamination: If you suspect contamination has occurred, perform a new cDNA synthesis reaction using your RNA template. Use new aliquots of SPRI beads for cDNA purification and equilibrate beads to room temperature before use. Note: RNA from certain cell types may have high copy numbers of specific transcripts. This will result in an abnormally high peak(s) or a family of peaks on the ds cDNA electropherogram. Always perform a negative (no RNA) control to discriminate between cell-specific gene expression patterns and possible contamination. The electropherogram shows a broad size distribution often with multiple small peaks. This is characteristic of a degraded RNA input sample. You may need to gather new RNA samples if you proceed with SMARTer Ultra low kits.