Question: Why is my total cDNA yield low?
Answer: Total cDNA yield is highly dependent on the cell type (RNA amount per cell; see notes below), cell preparation, and the cell number (note that the number of PCR cycles for cDNA amplification is adjusted as a function of the targeted cell number).
Low cDNA recovery may be the result of:
- Low total mRNA content per cell (cell-type dependent)
- Loading fewer cells than expected (dependent on cell counting technique and accuracy)
- Conditions used to pre-process cells (dissociation, FACS, any conditions that cause lysis, etc.)
- Cell viability (low cell viability can reduce transcriptional output as cells enter apoptosis)
- RT inhibitory contaminants (carryover additives or RNases/proteases from the cell suspension buffer/media)
- Sample clog
- Loss of cDNA during the emulsion breaking or clean-up steps
If you are consistently experiencing low cDNA yields, experiment with extra PCR cycles in the cDNA amplification and/or Sample Index PCR steps. However, keep in mind that the extra PCR cycles may increase PCR artifacts and may not increase final library complexity.
- Primary cells and cells of small cell size (i.e. PBMCs) generally have lower RNA content compared to immortalized cell lines or cancer cells.
- We have successfully generated libraries from cDNA yields as low as ~1-2 ng.
- A typical cDNA yield of 1000 RNA-poor cells is <5 ng/ul
- A typical cDNA yield of 1000 RNA-rich cells is >10 ng/ul
Products: Single Cell 3', VDJ