Table 2.
RNA Quality Guidelines by Analytical Platform
| Application | Criteria employed | Reference |
|---|---|---|
| RT-PCR | Amplification of a 60 bp product; DV200 > 30% | Personal Communication Dr. Betsou (IBBL, Luxembourg) |
| NGS | No RNA Integrity metric (RIN, DV200, etc.) useful in predicting if a library is successful. | Dr. Hoadley (UNC) |
| Predictors of success can come from libraries that yield >4 ng/μL concentration or MiSeq test runs, where goal is to see >10% of reads mapping to messenger RNA. | ||
| RNA capture required between 100 and 200 ng of total RNA derived from FFPE while Illumina Total RNA-Seq requires between 400 and 1000 ng. | ||
| Following library construction and Agilent/Caliper QC, the majority of fragments should be between 200 and 500 bp in length. Final library concentration should be >1 nM in at least 10 μL. | Dr. Andrew Mungall (BC Cancer) | |
| Ribosomal RNA depletion for RNA-Seq requires a minimum of 400 ng of total RNA input when quantified by Agilent Bioanalyzer/Caliper GX. Alternatively, 400 ng of total nucleic acid quantified by Qubit or Quant-iT can be used. | ||
| Sample Intake QC-DV200 should be >30%. RIN is not informative. | Dr. Harsha Doddapaneni and Dr. David Wheeler (Baylor College of Medicine Human Genome Sequencing Center) | |
| Library construction yields should be >3 ng with complementary DNA fragments between 100 and 1,500 bp when using between 50 and 100 ng RNA input. Greater input is required for samples with lower DV200 values. | ||
| Post-library capture should have >10 nM yield and devoid of primer dimers. | ||
| Microarray | >600 ng total RNA (by spectrophotometer); OD 260/280 ratio >1.5; 3′/5′ ratio <100 (as determined by TaqMan-based real time qRT-PCR of beta-actin using primers located 300 bp apart); Cy-dye incorporation >4.5 pmol/ng | Penland et al.26 |
| Ratio of real-time PCR amplicons of the 3′ to the 5′ end of beta-actin <20; Cycle threshold of the amplicon of the 5′ end of ACTB within seven cycles of the same quantity of universal control RNA | Roberts et al.27 | |
| Mean log ratio slope <0.15 due to the probe's distance from the 3′ end or its C-content in microarray hybridization | Duenwald et al.28 | |
| DASL | >100 ng RNA; A260/280 ratio >1.5; Rpll13a Ct values of <29 | Abramovitz et al.29 |
ACTB, beta-actin; DASL, cDNA-mediated Annealing, Selection, Extension and Ligation; RT-PCR, reverse transcription polymerase chain reaction.