Optimizing RNA Expression Analysis from Start to Finish to overcome major challenges ... Input...

Promega CorporationPromega Corporation

DNA Workflow

Marine Biological Laboratory

Mark BratzApplications Scientist, Promega Corporation

August 2016

2Promega CorporationPromega Corporation

Scientific Applications Support Mission

Realize customer driven applications of Promega technologies to

enhance the value of our technologies and strengthen our customer

relationships

Customer-specific testing & protocol development

Scientific Training

Custom field support (demos, seminars & experiments)


Presentation Outline

DNA Workflow• Purification• Quantitation

Key considerations at each step Ways to overcome major challenges Examples of challenging samples


DNA WorkflowEach Step Affects the Quality of the Final Data

Purify Quantify

PCR Amplify

qPCR

Sequencing

Microarray

Cloning


Downstream ApplicationsImportance of Input DNA Characteristics

PCR qPCR Sequencing Arrays

Quantity of DNA + + ++ +++

Integrity of DNA

+/-

Depending on amplicon

-

Typically small amplicons

+++

More important with longer read technologies, but many providers assume large fragments

++

Typically small fragments, but providers expect minimum fragment sizes

Lack of Inhibitors ++ ++ +++ ++

Accurate Quantitation ++ +++ +++ +++

Ultimately, sequencing data is dependent on the integrity and quality of the starting material


Purification:

Setting the Stage for Downstream Success

6


Purification Yield, Integrity & Purity are Critical to Success

Key Challenges

• Purifying sufficient DNA from:

• Low biomass samples

• Difficult samples

• Degraded samples

• DNA integrity

• Isolating pure DNA

• No enzyme inhibitors to affect downstream applications

• No contaminating RNA


DNA Purification TechnologiesAll Provide Advantages Depending on Specific Needs

ManualSmall

automated96 well manual

Automated


Purification, Manual Low Investment and Scalability are Attractive

Manual columns and scalable solution-based purification are attractive low-throughput options for standard or difficult samples

Advantages Reasons to Consider Other Options

Low initial investment vs. automation Greater throughput desired

Flexibility in sample processing Time constraints

Lower price per prep Error reduction

Minimal set up time

Many sample types supported: Blood, Tissue, FFPE, Plant…


DNA isolation from coffee beans

ReliaPrep gDNA Tissue Miniprep system


DNA isolation from coffee beans

DNA was isolated from coffee beans and was amplifiable using plant universal primers.


DNA Purification, Small Scale AutomationSmall Automated Systems Offer Major Benefits

Small, dedicated purification instruments allow individuals to automate purification and increase productivity


Minimal initial investment Not enough throughput to justify

Frees time for other activities Even greater throughput desired

Fewer purification errors Input sample volume incompatibility

Increases sample throughput

Maxwell® RSC: 5 minute setup – 30-45 minutes to extract 1-16 samples


Clinical

Research

AcademicApplied

Versatility of the Maxwell® RSC instrument


Maxwell® RSC Instrument


Microbes in water from Trunk River

Lysate

Purpose: Isolate DNA from microbes in Trunk River water using Maxwell RSC instrument. Experimental variables = Amount of water and homogenizing matrix.

Homogenization


Microbes in water from Trunk River

DNA was extracted using all homogenization matrices, but at different amounts. Sulfur-reducing bacteria was detected in the samples using qPCR.


DNA Purification, Manual 96 Well Vacuum Purification Increases Sample Throughput


Low initial investment 96 well processing can be tedious

High sample throughput Desire to reduce errors

Offers performance equal to spin columns

Staff time has become rate limiting

Greater throughput desired

Input sample volume incompatibility

96 well manual

The Wizard® SV 96 Genomic system can isolate gDNAfrom many sample types in less than 60 minutes


DNA Purification, Automated 96 Well Increases Laboratory Throughput and Lowers Costs


Increases laboratory productivity High initial cost

Aids in sample tracking Not enough throughput to justify

Increases consistency of results

Can automate many activities

Automated


Purification of DNA from food using automation

qPCR


Purification of DNA from food using automation

DNA was isolated from these food samples using an automated platform. The DNA was amplifiable using Universal Plant and Animal primers.


What sample types have challenged you?


Quantitation:

A Simple But Critical Step in Analysis

22


QuantitationA Simple But Critical Step in Analysis

Purify Quantify

PCR Amplify

qPCR

Sequencing

Microarray

Cloning


Challenges

Sensitivity

• effectively measuring small nucleic acid amounts

Accuracy

• affected by purity

• detection range

Specificity

• dsDNA vs ssDNA vs RNA

• human vs non-human

Quantify

Key Challenges Include Sensitivity, Accuracy, and Nucleic Acid Specificity


Three Common Methods of Quantitation Utilize UV Absorbance, Fluorescent Dyes and qPCR

UV Absorbance

•Spectrophotometer

•NanoDrop®/NanoVue™

Fluorescent Dye-based Quantitation

•Plate Reader

•Hand-held Instruments

Real-Time PCR


UV Absorbance Measures Different Components with Distinct Wavelengths

Wavelength Measurement

260nm

Amount of nucleic acid present in a sample

A260nm of 1.0 = 50µg/ml for dsDNA

40µg/ml for RNA

33µg/ml for ssDNA

280nm Amount of protein present in a sample

230nm Amount of other contaminants present in a sample

320nmAmount of light scattering components present in a sample; used for

background subtraction


Fluorescent Dye-based Quantitation is a More Sensitive Method

• Dye binds nucleic acid – the resulting conformation shift produces in fluorescence when excited

• Fluorescence is directly proportional to the amount of nucleic acid in the sample

• Higher signal = more nucleic acid present

• Unbound dye does not fluoresce

• Low background increases sensitivity

Incubate at room temp for 5 minutes

504nmExcitation

Emits @ 531nm

504nm

XUnbound dye

Easy Protocol: Add, Mix, Measure


Real-Time PCR (quantitative PCR, qPCR) Quantitation Involves Detection of Product at Each Cycle

What is end-point PCR?

The amount of amplified product is typically determined only after a set number of amplification cycles is completed

What is Real-Time PCR?

The amount of amplified product is measured after each PCR amplification cycle


Quantitation Activity

DNA Analysis Methods

Concentration Purity/ Contamination

Integrity Specificity –DNA/RNA

Sensitivity

Absorbance

Dye-Based Quantitation

qPCR


Equipment/Supplies Required

Relative Cost per Assay

Assay Time Hands-On Time Ability to Automate

Absorbance Spectrophotometer

Low <1min <1min Y


Fluorometer, Dye kits

Medium 10min 5min Y

qPCR Instrument, reaction comp.

High 1-2hr 15-30 min Y






Sensitivity

Absorbance Y


Y

qPCR Y






Low <1min <1min Y



Medium 10min 5min Y








Sensitivity

Absorbance Y Y


Y N

qPCR Y Y/N






Low <1min <1min Y



Medium 10min 5min Y




Quantitation of DNA by Absorbance Can Be Overestimated Due to Contaminants

Chemistry A Chemistry B

Large peak at 230nmNo peak at 230nm

gDNA Extraction from Matched Lung Tissue FFPE Slides

Absorbance is unreliable for Chemistry B – is qPCR a better choice?


Absorbance at 260nm May Not Be an Accurate Measure of Amplifiable Yield

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

breast breast colon colon lung lung

Chem A Chem B Chem A Chem B Chem A Chem B

ng/

ul

Tissue/method

Quantitation by Absorbance

0.0

1.0

2.0

3.0

4.0

5.0

6.0

breast colon lung

ng/

ul

Quantitation by Amplification

Chem A

Chem B

Large difference in quantitation• Absorbance at 260nm may

not be an accurate measure of amplifiable yield

• Absorbance and amplifiabilitymay correlate, but several other factors play a role


The NanoDrop® Instrument Often Overestimates the Amount of DNA in Solution

• Accurate quantitation is critical for many downstream applications

• Many FFPE tissue sections are small, and isolated DNA samples have concentrations well below the limit of detection of traditional spectrophotometric assays

• Even with highly purified DNA, the NanoDrop® consistently overestimates the amount of DNA in solution






Sensitivity

Absorbance Y Y


Y N

qPCR Y Y/N






Low <1min <1min Y



Medium 10min 5min Y








Sensitivity

Absorbance Y Y N


Y N N

qPCR Y Y/N Y/N






Low <1min <1min Y



Medium 10min 5min Y








Sensitivity

Absorbance Y Y N N


Y N N Y/N

qPCR Y Y/N Y/N Y






Low <1min <1min Y



Medium 10min 5min Y




A260 Absorbance is Not Specific and Cannot Distinguish Between dsDNA, RNA, or ssDNA

RNA

Sample A Sample B - + - + RNase

NanoDrop®

(ng/µl)

QuantiFluor™

dsDNA (ng/µl)

Sample A 213.5 158.0

Sample B 87.5 66.0

260nm reading represents total amount of all nucleic acid present in sample

Cannot distinguish between dsDNA, ssDNA, or RNA

DNA






Sensitivity

Absorbance Y Y N N


Y N N Y/N

qPCR Y Y/N Y/N Y






Low <1min <1min Y



Medium 10min 5min Y








Sensitivity

Absorbance Y Y N N Good10-12,000ng/ul


Y N N Y/N Better0.1-500ng

qPCR Y Y/N Y/N Y BestDepends






Low <1min <1min Y



Medium 10min 5min Y




Disadvantages of Absorbance Include Lack of Specificity, Overestimation, and Lack of Integrity Information

Lack of Specificity

• Cannot distinguish between dsDNA, RNA or ssDNA

• Nucleic acid contamination cannot be determined

Overestimation of nucleic acid concentration due to contaminants

• Many contaminants absorb at or around 260nm

No information on integrity

• Nucleotides and small nucleic acid fragments still contribute to the 260nm reading


Disadvantages of Fluorescent Dye-based Quantitation Include No Information on Purity or Integrity

Must create standards

No information on purity

• Separate dye-based quantification systems are available for ssDNA, RNA

and protein

No information on integrity

Lack of specificity with dyes

Fluorescent dyes are potentially hazardous


Disadvantages of Real-Time PCR Quantitation Include Expensive Equipment and Sensitivity to Inhibitors

ABI 7500 Real Time System

Bio-Rad MyiQ2

Requires specialized instrumentation

Higher cost compared to UV absorbance and fluorescent dye-based methods

Sensitivity to inhibitors

Bio-Rad QX100™ Droplet

Digital™ PCR System


Several Different Metrics Can Be Used to Predict the Likelihood of Success in Downstream Assays

• Quantitation by:

• Absorbance

• Fluorescent dye

• Amplification (qPCR)

• Purity by:

• Absorbance ratio

• Factors that impact quality:

• Purification chemistry contaminants

• Sample specific inhibitors

• Fragmentation

All are used to predict likelihood of success in downstream assays


NGS Workflow

The QuantiFluor® dsDNA dye system is designed to measure total double-stranded DNA concentration without regard for species, size, or amplifiability of the DNA, whereas a qPCR assay (84 bp target) is a human-specific qPCR test designed to measure amplifiable DNA. Samples with significantly lower qPCR quantitation results vs. fluorescent dye-based quantitation are indicative of degraded DNA.

Ratios of small to large amplicons as measured in the DNA QC assay are predictive of coverage uniformity and sequencing quality. For FFPE samples, a lower ratio of small (75 bp) to large (300 bp) amplicon target is indicative of less degradation of the DNA.

• Green = Amplicon ratios ≤47, coverage uniformity ≥93% • Yellow = Amplicon ratios 125-766, coverage uniformity 85-92% • Red = Amplicon ratios ≥ 1000, coverage uniformity ≤84%


Promega Application Lab at MBL

Contact: Mark Bratz

[email protected]

608-320-0697

July 31 – August 12

Office/Lab: Loeb 252

mailto:[email protected]


Questions

Optimizing RNA Expression Analysis from Start to Finish to overcome major challenges ... Input...

Documents

Transcript of Optimizing RNA Expression Analysis from Start to Finish to overcome major challenges ... Input...