rna integrity and quality – standardize rna quality control

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Title, Location, Date 1

RNA Integrity and Quality – Standardize RNA Quality Control Dr. Peter Porschewski MBA, QIAGEN GmbH

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IntroductionImportant considerations

Agenda

Critical factors for RNA preparation

RNA quality control

RNA integrity

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Important considerations

Challenges when working with RNA

Sample handling before extraction

Sample type and purification/extraction protocol

QC of RNA

Storage of RNA

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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

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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

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Agenda


RNA quality control

RNA integrity

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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

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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)

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Inactivation of RNases – purification of RNA from PBMCs

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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

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Agenda


RNA quality control

Determination of purity of RNA

Challenges of gDNA contamination

Stability of RNA after storage

RNA integrity

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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

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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?

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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!

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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.

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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

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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

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Agenda


RNA quality control




RNA integrity

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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

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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

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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)

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Agenda


RNA quality control




RNA integrity

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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

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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

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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

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Agenda


RNA quality control

RNA Integrity Score to assess RNA integrity

Importance of RNA integrity

QIAxpert for quantification

RNA integrity

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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

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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

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Agenda


RNA quality control




RNA integrity

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Six parameters are of prime relevance for RNA QC


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.

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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.

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Agenda


RNA quality control




RNA integrity

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Total RNA quality is assessed analysing the migration pattern


28S

18S

5S

18S/28S ratioSmears

Objectivity of the visual ratio estimation?Ratio is not always correlated to integrity!

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RIS and RIN objectively assess RNA integrity


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

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RNA Quality Control – RNA Integrity Score (RIS)

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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

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The gel images of QIAxcel and Agilent 2100 are comparable


QIA

xcel

Bio

anal

yzer

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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

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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)

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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

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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

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Total RNA analysis


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

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Total RNA analysis


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

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Q&A session

Thank you for your attention!

Questions?

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rna integrity and quality – standardize rna quality control

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