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Sample to Insight
Title, Location, Date 1
RNA Integrity and Quality – Standardize RNA Quality Control Dr. Peter Porschewski MBA, QIAGEN GmbH
Sample to Insight
2
IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
RNA integrity
Sample to Insight
Title, Location, Date 3
Important considerations
Challenges when working with RNA
Sample handling before extraction
Sample type and purification/extraction protocol
QC of RNA
Storage of RNA
Sample to Insight
4
Important considerations
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 controlTansportation
StorageExtractionPurificationStabilization 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
Sample to Insight
Title, Location, Date 5
Challenges for collection and stabilization
Sample materials
Sample type Collection Challenge for stabilization
Whole blood Blood collection tubesRBCs; protein content; cell membrane; volume
PlasmaBlood 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
TissuesDiff. 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
Sample to Insight
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IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
RNA integrity
Sample to Insight
Title, Location, Date 7
Critical factors for RNA preparation
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
Sample to Insight
Title, Location, Date 8
Critical factors for RNA preparation
Inactivation of RNases
RNA in homogenates is stable for several hours at RT
HS
HS
SH
SH
SHHS
HS
SH
Denatured, reduced ribonuclease
Native ribonuclease
GITC* andß-Mercaptoethanol
* Guanidine isothiocyanate
From Stryer: Biochemistry (3rd edition) DTT (1,4-dithiothreitol)ß-ME (β-Mercaptoethanol)
Sample to Insight
Title, Location, Date 9
Critical factors for RNA preparation
Inactivation of RNases – purification of RNA from PBMCs
Sample to Insight
Title, Location, Date 10
RNA quality control
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
Sample to Insight
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IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
Determination of purity of RNA
Challenges of gDNA contamination
Stability of RNA after storage
RNA integrity
Sample to Insight
Title, Location, Date 12
Determination of purity of RNA
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
Sample to Insight
Title, Location, Date 13
Determination of purity of RNA
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?
Sample to Insight
Title, Location, Date 14
260/230 Ratio
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!
Sample to Insight
Title, Location, Date 15
260/230 Ratio
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.
Sample to Insight
Title, Location, Date 16
Effect of phenol on UV absorbance
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
Sample to Insight
Title, Location, Date 17
Remaining phenol can inhibit downstream RT-PCR reactions
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
Sample to Insight
18
IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
Determination of purity of RNA
Challenges of gDNA contamination
Stability of RNA after storage
RNA integrity
Sample to Insight
Title, Location, Date 19
Challenges of gDNA contaminations
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
Sample to Insight
Title, Location, Date 20
Challenges of gDNA contaminations
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 RNAa. 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
Sample to Insight
Title, Location, Date 21
Challenges of gDNA contaminations
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 criteriaRNeasy
Plus Mini Kit
Supplier A Supplier B
Concentration RNA ng/µl 33,18 28,88 25,75Std. dev. 8,75 18,28 10,95
A 260/280 Ratio 2,1 2,01 2,17Std. dev. 0,04 0,09 0,12
Integrity RIN 9,6 9,3 9,2Std. dev. 0,16 0,43 0,66
Analysis of DNA depletion of total RNA purified from peripheral mononuclear cells (PBMCs)
Sample to Insight
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IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
Determination of purity of RNA
Challenges of gDNA contamination
Stability of RNA after storage
RNA integrity
Sample to Insight
Title, Location, Date 23
Stability of RNA eluates after storage
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
Sample to Insight
Title, Location, Date 24
Stability of RNA eluates after storage
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
Sample to Insight
Title, Location, Date 25
Stability of RNA eluates after storage
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
Sample to Insight
26
IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
RNA Integrity Score to assess RNA integrity
Importance of RNA integrity
QIAxpert for quantification
RNA integrity
Sample to Insight
Title, Location, Date 27
Importance of RNA integrity
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
Sample to Insight
Title, Location, Date 28
Importance of RNA integrity
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
Sample to Insight
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IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
RNA Integrity Score to assess RNA integrity
Importance of RNA integrity
QIAxpert for quantification
RNA integrity
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
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QIAxpert – excellent measurement accuracy
QIAxpert: lowest %CV value
Qubit: high mean variation
250 ng/µl reference RNA (Agilent Technologies)
AccuracyqPCR 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.
Sample to Insight
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QIAxpert – excellent linearity in RNA quantification
QIAxpert Nanodrop Qubit
0
200
400
600
800
1000
Nuc
leic
aci
ds (n
g/ul
)
0 200 400 600 800 1000Soll Konz (ng/µl)-1000
Excellent linearity Systematic overquantification
Underquantification turns in overquantification
Comparison of RNA linearity using different systems
LinearityHuman 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.
Sample to Insight
33
IntroductionImportant considerations
Agenda
Critical factors for RNA preparation
RNA quality control
RNA Integrity Score to assess RNA integrity
Importance of RNA integrity
QIAxpert for quantification
RNA integrity
Sample to Insight
Total RNA quality is assessed analysing the migration pattern
Title, Location, Date 34
28S
18S
5S
18S/28S ratioSmears
Objectivity of the visual ratio estimation?Ratio is not always correlated to integrity!
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
Sample to Insight
RNA Quality Control – RNA Integrity Score (RIS)
36
RIS: 9.5 RIS: 5.8
RIS: 3.6 superimposed
Lane Name RISA1 rat_liver _1 9.5
A7 rat_liver _4 5.8
A11 rat_liver _6 3.6
Comparison of different RNA quality
Sample to Insight
The gel images of QIAxcel and Agilent 2100 are comparable
Title, Location, Date 38
QIA
xcel
Bio
anal
yzer
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%
QIA
xcel
Bio
anal
yzer
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)
Sample to Insight
Title, Location, Date 42
ΔΔ
CT o
f actb
ΔΔ
CT
of hprt1
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
Sample to Insight
43
QIAxcel allows quality control of RNA using the RNA Integrity Score
Maximized efficiency and streamlining of your gene-expression workflow, QIAGEN SLAS 2015
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
TransportationStorage
ExtractionPurificationStabilization Molecular
analysis
Pre-analytical steps Analytical steps
1< RIS<10
Sample to Insight
Total RNA analysis
QIAxcel Advanced – Pure Excellence
RNeasy Mini Lipid Tissue Kit
QIAxcelAgilent Bioanalyzer
Qc M Qc M Qc M Qc M Qc
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
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
Sample to Insight
46
Q&A session
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.