How should I interpret unexpected peaks in my single cell gene expression cDNA trace?

The cDNA profile obtained from electrophoretic analysis typically shows a broad fragment size distribution ranging from approximately 400 to 9,000 bp, with an average fragment size of 1,200–1,600 bp. The actual size distribution may vary depending on the sample type and its gene expression profile.

Highly abundant transcripts may produce pronounced peaks at specific fragment sizes. These peaks can sometimes be associated with undesirable contributions, such as hemoglobin transcripts (~700 bp) from red blood cells or mitochondrial transcripts from stressed or damaged cells. However, pronounced peaks are not necessarily indicative of poor sample quality. In samples enriched for specific cell types, highly expressed transcripts may naturally generate distinct peaks. Examples include insulin transcripts in pancreatic beta cells.

Prominent peaks of  ≤300 bp typically indicate carryover of primer dimers from cDNA amplification. If these account for more than 5% of the total signal, an additional purification is recommended (refer to Singleron handbooks).