Question: Why is my Visium Spatial Gene Expression for FFPE Cq value for Cycle Number Determination so high?
Answer: Cq values are influenced by sample quality and workflow steps. Listed below are some potential root causes of high Cq values (>20 cycles).
Causes of high Cq values (>20 cycles):
- Use of sections with poor RNA quality (low DV200%)
- Use of sections thicker than 8 µm
- Use of tissues that are small in size
- Use of RNA-poor tissue
- Tissue detachment prior to Probe Release
- Insufficient Decrosslinking conditions (i.e. TE formulation/pH, temperature, or timing)
- Addition of undiluted Perm Enzyme B during the Pre-Hybridization step
- Incorrect Master Mix preparation (e.g. failure to add both LHS and RHS probes to the Hybridization Master Mix)
- Carryover of reagents or skipping wash steps
- Denaturation of ligated, extended probes with the incorrect concentration of KOH
- Failure to neutralize eluted Ligation product with 1M Tris-HCl, pH 7.0
- The use of third-party reagents not validated by 10x Genomics (e.g. untested staining dyes or alternatives to KAPA SYBR FAST qPCR Master Mix)
- Incorrect real-time controller set-up (e.g. program or SYBR detection; see: How do I interpret the qPCR plot to determine the Sample Index PCR cycle number?)
- Thermal cycler program (lid and/or block) incorrect
- Thermal cycler incompatible with Thermocycler Adaptor (see: Which thermal cyclers are compatible with Visium?)
- Reagents no longer within shelf-life or suboptimally stored
If you've gotten higher than expected Cq values (>20 cycles), please reach out to 10x Genomics Technical Support (firstname.lastname@example.org) for guidance.
Product: Visium for FFPE