RNA Integrity and Quality – Standardize RNA Quality Control
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This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
![Sample to Insight
Challenges of gDNA contaminations
Title, Location, Date 20
1. DNase treatment
a. On-membrane / on-bead
b. In solution (more efficient than on-membrane, esp. for large amounts of DNA
[mainly for sterical reasons])
2. Chemical separation of DNA and RNA
a. organic extraction – acid phenol / chloroform (e.g., QIAzol, …)
b. specific binding to solid matrix (e.g., RNeasy Plus, gDNA Eliminator)
3. DNA removal as part of cDNA synthesis protocol
a. e.g., QT Reverse Transcription kit
Combinations of different methods are possible
e.g., 1a. + 2a. or 2b. + 3., etc.
Different ways to eliminate genomic DNA](https://image.slidesharecdn.com/rnaintegrityquality-160106174212/85/RNA-Integrity-and-Quality-Standardize-RNA-Quality-Control-20-320.jpg)
RNA Integrity and Quality – Standardize RNA Quality Control
- 1. Sample to Insight RNA Integrity and Quality – Standardize RNA Quality Control Dr. Peter Porschewski MBA, QIAGEN GmbH Title, Location, Date 1
- 2. Sample to Insight IntroductionImportant considerations Agenda 2 Critical factors for RNA preparation RNA quality control RNA integrity
- 3. Sample to Insight Important considerations Title, Location, Date 3 Challenges when working with RNA Sample handling before extraction Sample type and purification/extraction protocol QC of RNA Storage of RNA
- 4. Sample to Insight Important considerations 4 Challenges when working with RNA Sample handling Time between collection and stabilization/lysis Changes in RNA transcript profile Stabilization Volume of stabilization solution Removal of stabilization reagent Sample degradation Transportation/Storage Storage temperature Sample degradation Extraction/Purification Lysis condition (tissue/cells) Sample size Sample degradation Contaminations Sample handling Quality control Tansportation Storage Extraction Purification Stabilization Molecular analysis Pre-analytical steps Analytical steps Quality control RNA quantification gDNA contamination RNA integrity Over-/underestimated yields Low integrity Molecular analysis Data interpretation Over-/underestimated gene expression level Low abundant transcripts Long transcripts Wrong detection of target gene
- 5. Sample to Insight Challenges for collection and stabilization Title, Location, Date 5 Sample materials Sample type Collection Challenge for stabilization Whole blood Blood collection tubes RBCs; protein content; cell membrane; volume Plasma Blood collection tubes + plasma separation Low amounts of free circulating analytes; protein content; volume Liquid Samples (e.g., urine, saliva) Various; depending on sample type Heterogeneity; cell membrane / cell wall; low amounts of free circulating analytes; protein content; volume Cellular samples (e.g., smears, swabs) Various; solid collection matrices Heterogeneity; cell membrane; cell lysis; collection matrices Tissues Diff. sizes (Surgical samples, biopsies); no standardization Compact structure; heterogeneity; sample size always limited Purified analytes (e.g., RNA/DNA) Various (e.g., tubes, plates); various buffers W/o additional extraction
- 6. Sample to Insight IntroductionImportant considerations Agenda 6 Critical factors for RNA preparation RNA quality control RNA integrity
- 7. Sample to Insight Critical factors for RNA preparation Title, Location, Date 7 Critical factors for RNA preparation RNA yield Efficient cell / tissue lysis mRNA content of total RNA is only 1–5% Purification of total RNA and small RNA (e.g., miRNA) Quality of the purified RNA: integrity and purity Quality of starting sample material Inactivation of RNases Copurification of potential inhibitors (e.g., for the RT step) Protein (nuclease) contamination gDNA contamination
- 8. Sample to Insight Critical factors for RNA preparation Title, Location, Date 8 Inactivation of RNases RNA in homogenates is stable for several hours at RT HS HS SH SH SH HS HS SH Denatured, reduced ribonuclease Native ribonuclease GITC* and ß-Mercaptoethanol * Guanidine isothiocyanate From Stryer: Biochemistry (3rd edition) DTT (1,4-dithiothreitol)ß-ME (β-Mercaptoethanol)
- 9. Sample to Insight Critical factors for RNA preparation Title, Location, Date 9 Inactivation of RNases – purification of RNA from PBMCs
- 10. Sample to Insight RNA quality control Title, Location, Date 10 Different aspects of RNA quality Purity Usually judged by OD ratios (260/280, 260/230) Absence of contaminants Absence of gDNA Stability of eluates Integrity – no degradation Usually judged by (capillary) gel electrophoresis (e.g., agarose gels, QIAxcel, Bioanalyzer, etc.) 3‘–5‘ ratio
- 11. Sample to Insight IntroductionImportant considerations Agenda 11 Critical factors for RNA preparation RNA quality control Determination of purity of RNA Challenges of gDNA contamination Stability of RNA after storage RNA integrity
- 12. Sample to Insight Determination of purity of RNA Title, Location, Date 12 OD ratios The ratio of the readings at 260 nm and 280 nm (A260/A280) and at 260 nm and 230 nm (A260/A230) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum, such as protein or organic solvents, salt, etc. 260/280 nm ratio: A260/A280 ratio is influenced considerably by pH Ideally around 1.8–2.1, at pH 7.5 (lower ratios at lower pH) For accurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A260/A280 ratio of 1.9–2.1 in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution used for dilution Low ratios commonly associated with protein contamination but sensitivity is rather low Low ratios vary often due to phenol contamination OD values close to background noise will also result in low ratios
- 13. Sample to Insight Determination of purity of RNA Title, Location, Date 13 OD ratios 260/230 nm ratio: The higher the better, there is consensus The expected 260/230 ratio for pure nucleic acid is often higher than the respective 260/280 ratio The expected 260/230 ratios are commonly in the range of 2.0–2.2 Low ratios are often associated with organic compounds or salts EDTA, carbohydrates and phenol have absorbance close to 230 nm Phenol absorbs also at ~270 nm Guanidine*HCl absorbs at ~230 nm Gunanidine thiocyanate absorbs at ~230 nm at moderate or low concentration – At very high concentrations GuSCN absorbs at 260 nm. What does it really mean?
- 14. Sample to Insight 260/230 Ratio Title, Location, Date 14 Thiocyanate absorbs very strongly around 220–230 nm GuSCN is present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures Based on our experience, the A260/A230 ratio of an RNA sample is strongly reduced when guanidine thiocyanate is present even at submillimolar concentrations Also, concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of real-time RT-PCR, even when using inhibitor sensitive PCR chemistries Low 260/230 ratio is mostly due to thiocyanate carryover!
- 15. Sample to Insight 260/230 Ratio Title, Location, Date 15 The most important factor is the amount of contaminant that is transferred to the downstream reaction, rather than the absorbance ratio Indicated concentrations are for eluates containing 50 ng/µl RNA 1 or 2 µl eluate used in 25 µl 1-step RT-PCR reaction for b-actin (inhibitory-sensitive chemistry) With 260/230 ratio around 1, still more than 1 order of magnitude before inhibition is observed Effect of guanidine salt concentration on the A260/A230 ratio and real-time RT-PCR.
- 16. Sample to Insight Effect of phenol on UV absorbance Title, Location, Date 16 Phenolic solutions absorbs in the UV both at 230 nm and with a maximum at ~270 nm Phenol contamination imitates higher RNA content of the sample
- 17. Sample to Insight Remaining phenol can inhibit downstream RT-PCR reactions Title, Location, Date 17 Total RNA was purified from rat muscle using the RNeasy Fibrous Tissue Mini Kit or Phenol- guanidine reagent qRT-PCR using the QuantiTect Probe RT-PCR Kit and primers / probes for c-jun CT values are shown with triplicates for each RNA amount Phenol remaining after RNA purification can reduce the efficiency of downstream applications
- 18. Sample to Insight IntroductionImportant considerations Agenda 18 Critical factors for RNA preparation RNA quality control Determination of purity of RNA Challenges of gDNA contamination Stability of RNA after storage RNA integrity
- 19. Sample to Insight Challenges of gDNA contaminations Title, Location, Date 19 Overestimation of RNA yield Overestimation of transcript abundance in qRT-PCR Especially rare or low-copy transcripts Higher background (e.g., microarrays, NGS) Consequences of DNA presence
- 20. Sample to Insight Challenges of gDNA contaminations Title, Location, Date 20 1. DNase treatment a. On-membrane / on-bead b. In solution (more efficient than on-membrane, esp. for large amounts of DNA [mainly for sterical reasons]) 2. Chemical separation of DNA and RNA a. organic extraction – acid phenol / chloroform (e.g., QIAzol, …) b. specific binding to solid matrix (e.g., RNeasy Plus, gDNA Eliminator) 3. DNA removal as part of cDNA synthesis protocol a. e.g., QT Reverse Transcription kit Combinations of different methods are possible e.g., 1a. + 2a. or 2b. + 3., etc. Different ways to eliminate genomic DNA
- 21. Sample to Insight Challenges of gDNA contaminations Title, Location, Date 21 The RNeasy Plus Mini Kit provided the most consistent RNA yields (lower variance) Total RNA was purified from 5 x 106 PBMC Real-time PCR amplification of the HOXD9 gene revealed approximately10-fold better elimination of gDNA contamination Quality assessment was done using the QIAxpert and Agilent Bioanalyzer Quality criteria RNeasy Plus Mini Kit Supplier A Supplier B Concentration RNA ng/µl 33,18 28,88 25,75 Std. dev. 8,75 18,28 10,95 A 260/280 Ratio 2,1 2,01 2,17 Std. dev. 0,04 0,09 0,12 Integrity RIN 9,6 9,3 9,2 Std. dev. 0,16 0,43 0,66 Analysis of DNA depletion of total RNA purified from peripheral mononuclear cells (PBMCs)
- 22. Sample to Insight IntroductionImportant considerations Agenda 22 Critical factors for RNA preparation RNA quality control Determination of purity of RNA Challenges of gDNA contamination Stability of RNA after storage RNA integrity
- 23. Sample to Insight Stability of RNA eluates after storage Title, Location, Date 23 t: 18 months at -20°C Ratio (28/18S): 1.7 RIN: 9.8 RNA from cultured cells: Jurkat Extracted with RNeasy Mini Kit t: 0 months Ratio (28/18S): 1.8 RIN: 9.8 RNA stability depends on purity
- 24. Sample to Insight Stability of RNA eluates after storage Title, Location, Date 24 t: 18 months at -20°C Ratio (28/18S): 1.6 RIN: 8.6 No significant change in RNA integrity, with RNA from cells or tissue RNA from tissue: rat spleen Extracted with RNeasy Mini Kit t: 0 months Ratio (28/18S): 1.7 RIN: 9.4 RNA stability depends on purity
- 25. Sample to Insight Stability of RNA eluates after storage Title, Location, Date 25 Storage of RNA Pure RNA can be stored at –80°C or – 20°C for prolonged periods Avoid repeated freeze-thaw cycles Store in aliquots Use low binding tubes Under this condition, no degradation of RNA is detectable even after 18 months
- 26. Sample to Insight IntroductionImportant considerations Agenda 26 Critical factors for RNA preparation RNA quality control RNA Integrity Score to assess RNA integrity Importance of RNA integrity QIAxpert for quantification RNA integrity
- 27. Sample to Insight Importance of RNA integrity Title, Location, Date 27 Integrity refers to how intact and undegraded the RNA is Important to obtain an accurate and quantitative measurement of gene expression at the moment of RNA extraction RNA integrity is limited mainly by the quality of the starting material 3‘/5‘ ratio: Signal from amplicons (RT-PCR) or capture probes (microarray) at different distances from 3’ end – after oligo-dT-based cDNA synthesis – may represent RNA quality
- 28. Sample to Insight Importance of RNA integrity Title, Location, Date 28 Check the intensity of the rRNA bands on an agarose gel or capillary electrophoresis Eukaryotic cells, the 28S should be double the intensity of the 18S band – Ratio of 28S:18S ribosomal RNA: Ideally 2:1, but difficult to determine. Bacterial cells – check the 23S in relation to the16S rRNA bands Note: Do not overload the gel as you will not get good clear separation If the ribosomal bands or peaks of a specific sample are not sharp, but appear as a smear towards smaller sized RNAs, it is likely that the sample suffered major degradation either before or during RNA purification. Integrity refers to how intact and undegraded the RNA is
- 29. Sample to Insight IntroductionImportant considerations Agenda 29 Critical factors for RNA preparation RNA quality control RNA Integrity Score to assess RNA integrity Importance of RNA integrity QIAxpert for quantification RNA integrity
- 30. Sample to Insight Six parameters are of prime relevance for RNA QC Title, Location, Date 30 QIAxcel Advanced and QIAxpert covers all RNA QC parameters There is no one-for-all solution QC Criteria Nanodrop Gels Qubit QIAxcel Advanced QIAxpert Protein contaminants (A260/280) Salts & other contaminants* (A260/A230) Yield () Degradation/ Sample integrity Size range Quantity of dsDNA vs. other NA
- 31. Sample to Insight QIAxpert – excellent measurement accuracy 31 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.
- 32. Sample to Insight QIAxpert – excellent linearity in RNA quantification 32 QIAxpert Nanodrop Qubit Excellent linearity Systematic overquantification Underquantification turns in overquantification 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.
- 33. Sample to Insight IntroductionImportant considerations Agenda 33 Critical factors for RNA preparation RNA quality control RNA Integrity Score to assess RNA integrity Importance of RNA integrity QIAxpert for quantification RNA integrity
- 34. Sample to Insight Total RNA quality is assessed analysing the migration pattern Title, Location, Date 34 28S 18S 5S 18S/28S ratio Smears Objectivity of the visual ratio estimation? Ratio is not always correlated to integrity!
- 35. Sample to Insight RIS and RIN objectively assess RNA integrity Title, Location, Date 35 RIS: RNA Integrity Score (QIAGEN) RIN: RNA Integrity Number (Agilent) Indicators reflecting RNA integrity Intended to predict the validity of downstream qPCR Frame of reference for RIS and RIN: Values range from 1 (highly degraded) to 10 (mostly intact) Analyze several different electropherograms’ parameters, including 28S and 18S peaks analysis Values between 7 and 10 are indicators of RNA quality suitable for downstream applications Depending on what is achievable with samples Allow comparison of sample, standardization and repeatability of experiments
- 36. Sample to Insight RNA Quality Control – RNA Integrity Score (RIS) 36 RIS: 9.5 RIS: 5.8 RIS: 3.6 superimposed Lane Name RIS A1 rat_liver _1 9.5 A7 rat_liver _4 5.8 A11 rat_liver _6 3.6 Comparison of different RNA quality
- 37. Sample to Insight The gel images of QIAxcel and Agilent 2100 are comparable Title, Location, Date 38 QIAxcelBioanalyzer
- 38. Sample to Insight RNA Quality Control – RNA Integrity Score (RIS) QIAxcel Advanced – Pure Excellence Comparable results to Agilent Bioanalyzer 2100 Lane Name RIS RIN A1 Jurkat_1 10.0 9.9 A2 Jurkat_1 10.0 9.9 A3 Jurkat_2 9.1 9.2 A4 Jurkat_2 9.1 9.2 A5 Jurkat_3 8.6 8.2 A6 Jurkat_3 8.9 8.2 A7 Jurkat_4 6.6 6.5 A8 Jurkat_4 6.7 6.5 A9 Jurkat_5 5.5 5.1 A6 Jurkat_3 5.6 5.1 A11 Jurkat_6 5.1 4.4 RIS RIN R² = 92.92% QIAxcelBioanalyzer
- 39. Sample to Insight Title, Location, Date 41 RNaseHeat UV light Depending on the degradation mechanism, the RNA has different electrophoretical behavior RIS is more robust than RIN to determine RNA integrity of RNA degraded by different methods High correlation between RIS and RIN values for heat and RNase III degraded RNA Lower correlation between RIN and RIS values for UV degraded RNA, due to high variation in RIN values, but similar at a decision level of RIN/RIS 7 Different RNA degradation methods result in different ranges of ΔΔCT values (1-log difference) RIS is more robust than RIN to evaluate suitability of RNA sample for qRT-PCR Unger C et al. (Electrophoresis. 2015 Sep;36(17):2072-81)
- 40. Sample to Insight Title, Location, Date 42 ΔΔCTofactbΔΔCTofhprt1 RIS for RNase degraded RNA The RIS is a good indicator to predict the outcome of gene expression experiments More information found by viewing the “Comparison of two RNA integrity indicators” webinar and reading the following publication: Unger C et al. (Electrophoresis. 2015 Sep;36(17):2072-81) RIS for heat degraded RNA RIS for UV degraded RNA
- 41. Sample to Insight QIAxcel allows quality control of RNA using the RNA Integrity Score Maximized efficiency and streamlining of your gene-expression workflow, QIAGEN SLAS 2015 43 Provides objective quality measurement for RNA samples Eliminates need for human interpretation and enables implementation of rigorous QC Gives highly reproducible results comparable to the Agilent Bioanalyzer Sample handling Quality control Transportation Storage Extraction Purification Stabilization Molecular analysis Pre-analytical steps Analytical steps 1< RIS<10
- 42. Sample to Insight Total RNA analysis QIAxcel Advanced – Pure Excellence RNeasy Mini Lipid Tissue Kit QIAxcelAgilent Bioanalyzer Qc M Qc MQc M Qc MQc M Qc M Hirn Qc M Qc M 25 mg brain tissue were disrupted with the QIAGEN TissueRuptor Storage on dry ice Average for RNA yield analysis 40 ng/µl
- 43. Sample to Insight Total RNA analysis QIAxcel Advanced – Pure Excellence RNeasy Mini Kit plus DNAse digestion Qc M Qc M Lunge Qc M Qc M QIAxcelAgilent Bioanalyzer 25 mg lung tissue disrupted with the QIAGEN TissueRuptor Storage on dry ice 1 µl of eluate analyzed Detection with Agilent or QIAxcel M
- 44. Sample to Insight Q&A session 46 Thank you for your attention! Questions? For up-to-date licensing information and product-specific disclaimers for QIAGEN products, 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.