Quality Control of RNA Samples — For Gene Expression Results You Can Rely On
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This slide deck covers the essentials of RNA QC as well as the differences between RIN and RIS.
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Quality Control of RNA Samples — For Gene Expression Results You Can Rely On
- 1. Sample to Insight 1 Dr. Pierre-Henri Ferdinand Global Product Management Pierre-henri.ferdinand@qiagen.com Quality Control of RNA samples — For gene-expression results you can rely on Webinar will start in a few minutes Quality Control of RNA samples
- 2. Sample to Insight Legal Disclaimer 2 QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor. Quality Control of RNA samples
- 3. Sample to Insight Outline Quality Control of RNA samples 3 Why is RNA quality control so important1 Main parameters for RNA QC2 Fast and reliable quality control3 Wrap-up4
- 4. Sample to Insight 4 http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165 “Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28 Billion/year spent on preclinical research that is not reproducible—in the United States alone.” Freedman LP, et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol. Quality Control of RNA samples
- 6. Sample to Insight 6 Non reproducibleReproducible biomedical research 50% • Culture of “publish or perish” • Inefficiencies in designing, conducting, and reporting of studies • Lack of standardization of sample quality for inter-lab reproducibility Some solutions for tackling the issue: • Implementing quality control procedures at key steps of laboratory workflows to help standardizing the parameters of the samples, and thus the quality of the data generated. • Combining lab automation with quality chemistries to enhance reproducibility of experiments, bringing confidence in data interpretation. Quality Control of RNA samples
- 7. Sample to Insight 7 QC emphasizes on gaining insight into a sample quality and assessing its suitability for downstream applications and its potential for generating reliable results. QC plays an important role when it comes to developing routine procedures or troubleshooting an experiment. µg ? Purity? Quantity? Integrity? Size? Are these parameters in the range of my experiment’s requirements for generating high-quality results? RNA Quality? What is RNA sample QC? Quality Control of RNA samples
- 8. Sample to Insight 8 Why is sample QC so important? Risk of failure Molecular biology workflows are complex, error- and failure-prone multi-step procedures A lot of things can potentially go wrong during workflows RNA can be unstable: sensitive to heat, radiations and nucleases Confidence in results At the end, only the quality of the final result matters The quality of the final results is highly influenced by quality of samples Cost aspects Downstream applications get more sensitive but not necessarily more robust to variations in samples quality Failure means waste of time and resources Price per sample Time qPCR 1.5 $US 4-5 hrs Sanger Sequencing 5-6 $US 4-6 hrs Next-Gen Sequencing >200 $US(5plex) 2-3 working days Affymetrix GeneChip Analysis 500-800 $US/sample > 2 working days Quality Control of RNA samples
- 9. Sample to Insight 9 Sample collection/ stabilization RNA purification, cDNA & cRNA synthesis Quantification, Normalization, Fragmentation Array Hybridization and scanning Data analysis & interpretation Gene expression Microarrays QC QC Sample collection/ stabilization RNA purification Amplification Analysis Data analysis & interpretation Gene expression RT-PCR QC Sample collection/ stabilization DNA/RNA purification NGS Library Prep Sequencing Data analysis & interpretation RNA-Seq QC QC RNA Quality Control is recommended in Gene expression workflows RNA Quality Control after purification Quality Control of RNA samples
- 10. Sample to Insight Outline Quality Control of RNA samples 10 Why is RNA quality control so important1 Main parameters for RNA QC2 Fast and reliable quality control3 Wrap-up4
- 11. Sample to Insight Six parameters of prime relevance for sample Quality Control Quality Control of RNA samples 11 QC Criteria UV spectro. Gels Dye-based spectro. CE / µ- fluidic UV/VIS spectro. Protein contaminants (A260/280) Salts & other contaminants (A260/230) Quantity of dsDNA vs. other NA Yield () Degradation/ Size distribution () Sizing There is no one-for-all solution – 2 technologies cover all sample QC needs purity quantity integrity
- 12. Sample to Insight Main RNA quality parameters Quality Control of RNA samples 12 Purity Quantity Integrity What is the yield of my extraction? How much RNA is in the tube and at what concentration? Will I have enough material? When the concentration of NA is overestimated, the input amount will be too low. This leads to weak amplification and weak signal strength. When the concentration of DNA is underestimated the input amount will be too high and can lead to overuse of precious samples, increased concentration of inhibitors, false priming, unfavorable ratios, etc. What’s really in my tube? Could contamination or impurities interfere with my assay? Phenol, ethanol, salts, etc. can jeopardize sensitivity and efficiency of downstream enzymatic reactions. Large amounts of unwanted ssDNA or dsDNA template lead to overestimation of the molecule of interest and interferes with downstream applications and introduce bias in quantitative assays. Does my RNA sample have the correct size distribution? Has RNA been degraded? RNA samples of poor integrity can affect PCR or sequencing reactions and lead to errors in replication and/or lower yields e.g. irrelevant CT values. Degraded samples can produce false negative results.
- 13. Sample to Insight Quality Control of RNA samples 13 • RNA QC requirements vary depending on downstream applications: RT-PCR, qRT-PCR, Microarray or RNA-Seq • Small deviations in key sample parameters can have potential impact on final results Implementation of QC procedures with relevant pass-criteria and ensuring RNA samples quality parameters are within acceptable range are essential to maximize success of experiments, thus saving time, money and peace of mind Highest quality results are only achieved from quality samples Only quality results are worth sharing
- 14. Sample to Insight Key quality indicators Quality Control of RNA samples 14 QC Criteria UV spectro. Gels Dye-based spectro. CE / µ- fluidic UV/VIS spectro. Protein contaminants (A260/280) Salts & other contaminants (A260/230) Quantity of dsDNA vs. other NA Yield () Degradation/ Size distribution () Sizing There is no one-for-all solution purity quantity integrity
- 15. Sample to Insight Key quality indicators Quality Control of RNA samples 15 QC Criteria UV spectro. Gels Dye-based spectro. QIAxcel Advanced QIAxpert Protein contaminants (A260/280) Salts & other contaminants (A260/230) Quantity of dsDNA vs. other NA Yield () Degradation/ Size distribution () Sizing purity quantity integrity There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!
- 16. Sample to Insight Outline Quality Control of RNA samples 16 Why is RNA quality control so important1 Main parameters for RNA QC2 Fast and reliable quality control3 Wrap-up4
- 17. Sample to Insight How does QIAGEN helps you achieving reliable QC? Quality Control of RNA samples 17 Quantity & Purity Integrity & size distribution QIAxpert QIAxcel Advanced Let lab automation work for you and do the job they are excellent at: Delivering reliable and reproducible results Perform accurate measurements, keep track of your sample quality, standardize your experiments and increase reproducibility of your results. Increase your lab productivity: automation frees your hands and your mind!
- 18. Sample to Insight Quality Control of RNA samples 18 Sample collection/ stabilization RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation QCQC Quantity & Purity Integrity & size distribution QIAxpert QIAxcel Advanced
- 19. Sample to Insight QIAxpert uses innovative microfluidic sample carrier 19 Key features: 1 2 3 4 5 1 2 3 4 5 Sample loading well Capillaries Microcuvette Overflow Vent hole for vacuum Only 2 µl of sample required DNA range: 1.5 ng/μl up to 2000 ng/μl dsDNA (A260) Fast analysis, up to 16 samples in less than 2 minutes Algorithm can unmixe sample’s spectra to differentiate contribution of different molecules. QIAxpert is a high-speed spectrophotometer for DNA, RNA and protein analysis. It is fast, accurate and sensitive. Quality Control of RNA samples
- 20. Sample to Insight Quality Control of RNA samples 20 Classic spectrophotometry A230: impurities A280: proteins A260: nucleic acids
- 21. Sample to Insight Quality Control of RNA samples 21 1. Absorbance measurement (and background correction) 2. Content profiling of the measured spectrum by fitting of reference spectra into • Specific DNA or RNA spectrum • Impurities spectrum • Residue spectrum 3. Quality control • Impurities spectrum • Residue spectrum • Backround spectrum • A260/A280 ratio for protein contamination QIAxpert – Unique spectral content profiling protocols Quantity of RNA you think you have Quantity of RNA you actually have
- 22. Sample to Insight QIAxpert – Excellent measurement accuracy 22 QIAxpert: lowest %CV value Qubit: high mean variation 250 ng/µl reference RNA (Agilent Technologies) Accuracy qPCR Human Reference Total RNA (Agilent Technologies) was diluted to 250 ng/µL (dilution from original solution in H2O). A total of 40 replicates were measured, each on the QIAxpert (RNA260 app), on a Nanodrop 8000, and the Qubit system. Quality Control of RNA samples
- 23. Sample to Insight QIAxpert – Excellent linearity in RNA quantification 23 QIAxpert Nanodrop Qubit Excellent linearity Systematic overquantification Systematic underquantification Comparison of RNA linearity using different systems Linearity Human Reference RNA (Agilent) was diluted to 1000 ng/µl, 500 ng/µl, 100 ng/µl, 50 ng/µl, 10 ng/µl, 5 ng/µl, and 1.5 ng/µl. A total of 5 replicates of each dilution were measured using the QIAxpert system, a Nanodrop 8000, and the Qubit. Data shown for the QIAxpert reflects total NA measured with the RNeasy app. Quality Control of RNA samples
- 24. Sample to Insight With QIAxpert – tell RNA from DNA and other contaminants without a dye 24 High-speed microfluidics • No drop & clean action required, no cross contamination • Evaporation-safe for 2h • Flexible input & up to 16 samples in <2 min Comprehensive Export • Reports can be viewed on any browser • USB and Network data output • No extra software or computer required Easy interaction • Inbuilt touchscreen • Simple GUI • Variety of pre-installed protocols Spectral content profiling • Differentiation between molecules • Quantify and subtract identified impurities • Give best insight into samples’ quality Quality Control of RNA samples
- 25. Sample to Insight Quantity & Purity ! (without dye) QIAxpert – Unique spectral content profiling protocols 25 Classic spectrophotometry Quantity Spectral Content Profiling For internal use only • DNA or RNA spectrum • Impurities spectrum • Residue spectrum Quality Control of RNA samples
- 26. Sample to Insight Quality Control of RNA samples 26 Sample collection/ stabilization RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation QCQC Quantity & Purity Integrity & size distribution QIAxpert QIAxcel Advanced
- 27. Sample to Insight QIAxcel Advanced allows separation, detection and analysis of NA 27 QIAxcel Advanced Fully automated DNA and RNA analysis Fast processing: 12 samples in 8 – 13 min Up to 96 samples per run Up to twenty 96-well plates with QIAxcel HT High resolution down to 3 – 5 bp up to 500 bp Safety and convenience with ready-to-use gel cartridges Digital data output QIAxcel Advanced is an automated capillary electrophoresis system for DNA and RNA analysis. QIAxcel Advanced allows analysis of gDNA, RNA and NGS libraries Quality Control of RNA samples
- 28. Sample to Insight QIAxcel Advanced capillary electrophoresis principle 28Quality Control of RNA samples • Reusable 12-capillary cartridges • Fast processing: 12 samples in 8-13 min • Detection limit of 0.1 ng/µl • Sample consumption < 0.1µl
- 29. Sample to Insight How does it work? Place the gel cartridge into the instrument Load samples (tube strips or 96-well plates), buffers, and markers Select the Process Profile of your choice... and GO! Look at the results in real-time on the screen and report data just a few minutes later Operating the QIAxcel Advanced system: Quality Control of RNA samples 29
- 30. Sample to Insight Total RNA quality is assessed by analysing the migration pattern 30 28S 18S 5S 18S/28S ratio Smears Objectivity of the visual ratio estimation? Ratio is not always correlated to integrity! Quality Control of RNA samples
- 31. Sample to Insight RNA Quality Control - RNA Integrity Score (RIS) 31 A1 RIS: 9.5 A7 RIS: 5.8 A11 RIS: 3.6 Superimposed view Lane Name RIS A1 rat_liver _1 9.5 A7 rat_liver _4 5.8 A11 rat_liver _6 3.6 Quality Control of RNA samples RIS 10 1 RNA Integrity
- 32. Sample to Insight 32 The gel images of QIAxcel and Agilent 2100 are comparable QIAxcelBioanalyzer Quality Control of RNA samples
- 33. Sample to Insight 33 More information at lower molecular weight on the QIAxcelQIAxcelBioanalyzer
- 34. Sample to Insight RIS and RIN can objectively assess RNA integrity 34 RIS : RNA Integrity Score, by QIAGEN RIN : RNA Integrity Number, by Agilent • Indicators reflecting RNA integrity • Intended to predict the validity of downstream qPCR Frame of reference of RIS and RIN: • Values range from 1 (highly degraded) to 10 (mostly intact) • Analyse several different electropherogram’s parameters, including 28S and 18S peaks analysis • Values between 7 and 10 (depending of what is achievable with samples) are indicators of RNA quality suitable for downstream applications • Allow comparison of sample, standardization and repeatability of experiments Quality Control of RNA samples
- 35. Sample to Insight RIS is more robust than RIN for assessing RNA integrity 35 • Depending on the degradation mechanism (heat, nuclease or UV), total RNA samples have different electrophoretic behavior • Different RNA degradation methods result in different ranges of ΔΔCT values for identical RIS/RIN values • “Overall, RIS was more robust than RIN for assessing RNA integrity” Unger et al, 2015 http://www.ncbi.nlm.nih.gov/pubmed/25998866 www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement Learn more about RNA quality control from our RNA QC webinar. Watch it at www.qiagen.com.
- 36. Sample to Insight Outline Quality Control of RNA samples 36 Why is RNA quality control so important1 Main parameters for RNA QC2 Fast and reliable quality control3 Wrap-up4
- 37. Sample to Insight Sample Insight Sample collection/ stabilization DNA/RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation Sample QC must be part of molecular biology workflows, not optional steps on top. QC brings value to workflow and can save cost, time and peace of mind QC QC Quality Control of RNA samples
- 38. Sample to Insight Key quality indicators Quality Control of RNA samples 38 QC Criteria UV spectro. Gels Dye-based spectro CE / µ- fluidic UV/VIS spectro. Protein contaminants (A260/280) Salts & other contaminants (A260/230) Quantity of dsDNA vs. other NA Yield () Degradation/ Size distribution () Sizing There is no one-for-all solution purity quantity integrity
- 39. Sample to Insight Key quality indicators Quality Control of RNA samples 39 QC Criteria UV spectro. Gels Dye-based spectro. QIAxcel Advanced QIAxpert Protein contaminants (A260/280) Salts & other contaminants (A260/230) Quantity of dsDNA vs. other NA Yield () Degradation/ Size distribution () Sizing purity quantity integrity There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!
- 40. Sample to Insight Size distribution analysis Quantity & Purity QIAGEN covers all your needs for sample Quality Control 40 Spectral Content Profiling For internal use only Integrity QIAxpert QIAxcel Advanced Quality Control of RNA samples Success and reproducibility of assays ; relevance of quantitative experiments
- 41. Sample to Insight Sample QC can save you time, money, and peace of mind Quality Control of RNA samples 41 Application Price per sample Time Downstream application qPCR 1.5 $US 4-5 hrs Sanger Sequencing 5-6 $US 4-6 hrs Next-Gen Sequencing >200 $US(5plex) 2-3 working days Affymetrix GeneChip Analysis 500-800 $US/sample > 2 working days Sample Quality Control QIAxpert <1$US 90 seconds (16 samples in parallel) QIAxcel Advanced <1$US 3-10 minutes (12 samples in parallel) Purity & quantity Integrity QC gives insights into sample suitability for downstream application • Brings confidence in data analysis and interpretation, troubleshooting • Saves time, money, and peace of mind by excluding samples of poor quality “Save time, money, and peace of mind only dealing with the samples you deserve: the ones of highest quality”
- 42. Sample to Insight How does QIAGEN helps you achieving reliable QC? Quality Control of RNA samples 42 Quantity & Purity Integrity & size distribution QIAxpert QIAxcel Advanced Let lab automation work for you and do the job they are excellent at: Delivering reliable and reproducible results Perform accurate measurements, keep track of your sample quality, standardize your experiments and increase reproducibility of your results. Increase your lab productivity: automation frees your hands and your mind!
- 43. Sample to Insight Quality Control of RNA samples 43 Quantity & Purity Integrity & size distribution QIAxpert QIAxcel Advanced More info on: QIAGEN.com Discover how these instruments can help you improve the reproducibility of your experiments and achieve higher quality results with our interactive online demo tools and request a free demo in your lab!
- 44. Sample to Insight 44 Questions? Thank you for your attention! Pierre-Henri Ferdinand Global Product Management Pierre-henri.ferdinand@qiagen.com Learn more about nucleic acids QC on: qiagen.com/QCSolutions Quality Control of RNA samples