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 such as EDTA or Mg2+, or RNases/proteases from the cell suspension buffer/media)
- Sample clog
- Loss of cDNA during the emulsion breaking or clean-up steps
- Resuspension of TSO in the incorrect buffer (regular TE instead of low TE, for example)
If you are consistently experiencing low cDNA yields and suspect your samples have low RNA content (small cells, nuclei), experiment with adding an extra 1-2 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 Gene Expression, Single Cell Immune Profiling