Table 3.
Guidelines recommended for the study of circulating lncRNAs as biomarkers for cancer diagnosis, based on troubleshooting performed by previous works.
| Step | Recommended | Reason | Reference |
|---|---|---|---|
| Patient selection | Exclude patients with inflammation | Higher / different levels of white blood cells associated with inflammation may impact levels of circulating RNAs upon cytolysis | 198,199 |
| Recruit patients with same gender, age and race | Minimize variation in lncRNA levels due to possible inter-individual confounding factors (such as SNPs, CNV, etc.) | 63 | |
| May include questionnaire about diet and lifestyle | Diet and lifestyle (alcohol consumption, smoking) can affect lncRNA levels | 200,201 | |
| Blood sample preparation | Prepare serum or plasma. Discard cellular fraction | Cellular fraction of blood may contain different levels of blood cells which in return may impact levels of circulating RNAs upon cytolysis | 199,202 |
| Strict standard operating procedures when preparing serum/plasma | Minimize variations in circulating RNAs due to sample preparation. Avoid hemolysis. | 202 | |
| Measure A414, A541, A576 | Assess for hemolyzed samples | 69 | |
| RNA extraction | Use kits compatible with liquid samples | Enable extraction of circulating lncRNAs from plasma or serum samples | Kit manufacturers |
| Use kits combining both solid (filter) and liquid phase (organic) extraction | Maximize extraction of circulating lncRNAs from plasma or serum samples | 17,24,42 | |
| Use as much plasma/serum as possible | Maximize RNA yield after extraction | Our recommendation | |
| Reverse Transcription | Use same volume of RNA extracts | Allow maximum RNA input for Reverse Transcription | Our recommendation |
| qPCR (relative quantification with ΔΔCt method) | Test several reference genes. Carefully choose best reference gene(s) using NormFinder, RefFinder or Genorm algorithms. Most popular: GAPDH, beta-actin, 18 S To avoid: RPLPO, GUSB, HPRT | The right reference gene is needed for accurate relative quantification using ΔΔCt method. GAPDH, beta-actin, 18 S present in large quantities in blood. RPLPO levels inconsistent in blood GUSB, HPRT levels too low in blood | 3,25,41,42,47,203 |
| Careful in interpretation of data when using spike-in controls | Spike-in controls do not account for variations in lncRNA concentrations in blood-derived samples prior to RNA extraction step | 180 | |
| Measure transcript levels of MB, NGB, CYGB genes | Assess for contamination from red blood cells | 69 | |
| Measure transcript levels of APOE, CD68, CD2, CD3 genes | Assess for contamination from white blood cells | 69 |
Information reported includes step of the analysis, actual recommendation, reason for the recommendation and related literature reference.