| Observation | Possible Cause | Recommended Action |
|---|---|---|
| The yield of PCR products for RNA samples is low but is good for Positive Control RNA sample — or — Low yield of PCR products for both experimental and Positive Control RNA samples |
Error in sorting cells, or cellular RNA is degraded | Optimize sorting conditions, sort only viable cells using dead-cell staining protocol, and add RNAse inhibitor (e.g., 1μl/well) in Hybridization Master mix before sorting. |
| Low RNA content in sorted cells | Increase the number of amplification cycles in the First PCR with anchor primer or 2nd PCR with Indexed Primers. | |
| RT step is not optimal | Please check for procedural mistakes. Make master mixes where possible. For individual reaction setups, make sure that the correct volume of RT-EXT Buffer and RT enzyme is added into each reaction. | |
| PCR cycling conditions are not optimal | Increase PCR amplification cycles. Ensure proper dispensing and mixing of viscous components at each step. |
| Observation | Possible Cause | Recommended Action |
|---|---|---|
| Low Cluster Density | Inefficient purification from low molecular weight RNA products or low yield of correct target PCR products | Re-purify PCR products using different technology (e.g. QIAGEN QIAquick PCR Purification Kit), and/or increase the concentration of pooled Indexed Library products in the cluster generation step. |
| Observation | Possible Cause | Recommended Action |
|---|---|---|
| Yields of PCR products are significantly higher than Positive Control | RNA content in sorted cells is too high | Decrease number of PCR cycles in 1st or 2nd PCR steps |
| Low fluorescence readings on Illumina sequencer — or — Lower than expected number of on-target reads — or — High mutation rate in the Index reads |
PCR primers from amplified indexed library quantification step (Quantify and Combine Samples for NGS) were not removed | Incubate Amplified Indexed Library with Primer Removal Buffer and Primer Removal Enzyme and repeat purification and quantification steps. |
| Lower than expected number of on-target reads | Yield of amplified products is too low or PCR cycle number too low | Increase the number of PCR cycles in the 1st PCR or 2nd PCR step. |
| Amplified Indexed Library has off-target contaminations | Re-purify pooled Amplified Indexed Library in agarose gel (200bp-700bp products) and repeat quantification and NGS step. | |
| Uneven representation of Indexed Libraries | Inaccurate Indexed Library quantification | Check that you correctly calculated the molar concentration of each indexed library sample. |
| Inaccurate Indexed Library mixing | Re-quantify the indexed library samples and mix them in equimolar amounts. | |
| Inconsistent library yields from replicate or similar sorted cell samples | Sample evaporation in thermal cycler or mistakes in adding correct volume of reagents in different wells | Seal 96-well plates well with an Adhesive Film Applicator and use a Compression Pad. Visually control the volume of reagents aliquoted in different wells/test tubes and similar volumes in wells/test tubes after incubation steps. |
Last modified:
18 October 2024
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