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.

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.