RNA Quality Control - Comparing Different RNA Quality Indicators

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Transcript of RNA Quality Control - Comparing Different RNA Quality Indicators

Legal Disclaimer

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Dr. Pierre-Henri FerdinandGlobal Product Management Pierre-henri.ferdinand@qiagen.com

Quality Control of RNA samplesRNA quality control comparing different RNA quality indicators

Sample to InsightLegal Disclaimer2QIAGEN 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

Sample to Insight

OutlineQuality Control of RNA samples3

Why is RNA quality control so important1Main parameters for RNA QC2Fast and reliable quality control3Comparison of RIN and RIS4Wrap up5

Sample to Insight

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http://www.sciencemag.org/news/2015/06/study-claims-28-billion-year-spent-irreproducible-biomedical-researchhttp://www.nature.com/news/reproducibility-1.17552http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165

Quality Control of RNA samples

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Non reproducibleReproducible biomedical research50%

Culture of publish or perishInefficiencies in designing, conducting, and reporting of studiesLack 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

Sample to Insight6QC 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.

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Purity?Quantity?Integrity?Size?Are these parameters in the range of my experiments requirements for generating high-quality results? RNA Quality?What is RNA sample QC?Quality Control of RNA samples

Sample to Insight7Why is sample QC so important?Risk of failureMolecular biology workflows are complex, error- and failure-prone multi-step proceduresA lot of things can potentially go wrong during workflowsRNA can be unstable: sensitive to heat, radiations and nucleases

Confidence in resultsAt the end, only the quality of the final result mattersThe quality of the final results is highly influenced by quality of samples

Cost aspectsDownstream applications get more sensitive but not necessarily more robust to variations in samples qualityFailure means waste of time and resources

Price per sampleTimeqPCR1.5 $US4-5 hrsSanger Sequencing5-6 $US4-6 hrsNext-Gen Sequencing>200 $US(5plex)2-3 working daysAffymetrix GeneChip Analysis500-800 $US/sample> 2 working days

Quality Control of RNA samples

Sample to Insight8Sample collection/ stabilizationRNA purification, cDNA & cRNA synthesisQuantification, Normalization, FragmentationArray Hybridization and scanningData analysis & interpretation

Gene expression MicroarraysQCQCSample collection/ stabilizationRNA purificationAmplificationAnalysisData analysis & interpretation

Gene expression RT-PCRQCSample collection/ stabilizationDNA/RNA purificationNGS Library PrepSequencingData analysis & interpretation

RNA-Seq QCQCRNA Quality Control is recommended in Gene expression workflows

RNA samples Quality Control and standardizationQuality Control of RNA samples

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OutlineQuality Control of RNA samples9

Why is RNA quality control so important1Main parameters for RNA QC2Fast and reliable quality control3Comparison of RIN and RIS4Wrap up5

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Six parameters of prime relevance for sample Quality ControlQuality Control of RNA samples10QC CriteriaUV spectro.GelsDye-based spectro.CE / -fluidicUV/VISspectro.Protein contaminants(A260/280)Salts & other contaminants(A260/230)Quantity of dsDNA vs. other NAYield()Degradation/Size distribution()Sizing

There is no one-for-all solution 2 technologies cover all sample QC needs

purityquantity

integrity

Sample to InsightMain RNA quality parametersQuality Control of RNA samples11

Purity QuantityIntegrityWhat 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.Whats 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.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.

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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 resultsImplementation 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

Sample to InsightKey quality indicatorsQuality Control of RNA samples13QC CriteriaUV spectro.GelsDye-based spectro.CE / -fluidicUV/VISspectro.Protein contaminants(A260/280)Salts & other contaminants(A260/230)Quantity of dsDNA vs. other NAYield()Degradation/Size distribution()Sizing

There is no one-for-all solution

purityquantity

integrity

Sample to InsightKey quality indicatorsQuality Control of RNA samples14QC CriteriaUV spectro.GelsDye-based spectro.QIAxcel AdvancedQIAxpertProtein contaminants(A260/280)Salts & other contaminants(A260/230)Quantity of dsDNA vs. other NAYield()Degradation/Size distribution()Sizing

purityquantity

integrityThere is no one-for-all solution QIAGEN covers all your needs for sample Quality Control!

Sample to Insight

OutlineQuality Control of RNA samples15

Why is RNA quality control so important1Main parameters for RNA QC2Fast and reliable quality control3Comparison of RIN and RIS4Wrap up5

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Quality Control of RNA samples16Sample collection/ stabilizationRNA purificationAmplification/ NGS Library PrepAnalysisData analysis & interpretation

QCQC

Quantity & PurityIntegrity & size distributionQIAxpertQIAxcel Advanced

Sample to InsightQIAxpert uses innovative microfluidic sample carrier 17Key features:

1234512345Sample loading wellCapillariesMicrocuvetteOverflowVent hole for vacuum

Only 2 l of sample required

DNA range: 1.5 ng/l up to 2000 ng/l dsDNA (A260)

Fast analysis, up to 16 samples in less than 2 minutes

Algorithm can unmixe samples 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

Sample to InsightQuality Control of RNA samples18

Classic spectrophotometryA230: impuritiesA280: proteinsA260: nucleic acids

Sample to InsightQuality Control of RNA samples19

Absorbance measurement (and background correction)

Content profiling of the measured spectrum by fitting of reference spectra into

Specific DNA or RNA spectrumImpurities spectrum Residue spectrum

Quality controlImpurities spectrumResidue spectrum Backround spectrumA260/A280 ratio for protein contamination

QIAxpert Unique spectral content profiling protocolsQuantity of DNA or RNA you think you have Quantity of DNA or RNA you actually have

Sample to InsightWith QIAxpert tell RNA from DNA and other contaminants without a dye20

High-speed microfluidics No drop & clean action required, no cross contamination Evaporation-safe for 2h Flexible input & up to 16 samples in RINRIS>RINRIS>RINRNase digestionHeat degradationUV-lightexposureCorrelation Ct and RIS values R= 89,7%R= 89,6%R= 97,4%Correlation Ct and RIN values R= 89,6%R= 87,6%R= 93,1%

Figure: the correlation of Ct-values with RIS and RIN values Calculated by a linear regression analysis with Minitab 16, to study whether RIS and RIN could predict the outcome of a qPCR. Fitted line plots with regression line and adjusted R2 values of RIS and RIN against Ct-values of ACTBQuality Control of RNA samples

Sample to Insight

Heat