Prepared by Michaela Bowden Ph.D
Automated TruSeq RNA Sample Preparation from FFPE tissue specimens utilizing the Biomek
FXP Liquid Handler
Michaela Bowden PhD Dana Farber Cancer Institute
21st March 2013
Prepared by Michaela Bowden Ph.D
Dana Farber Cancer Institute • Founded in 1947 for Sidney Farber MD
• Mission: to offer cancer patients the best treatment available today, while developing tomorrow’s cures through research
• DFCI holds > 400 NIH and NCI grants and is one of the most successful institutions in the country for securing cancer-related funding due to the quality of research being conducted there
• DFCI, as a non-profit organization, also benefits from the generosity of individuals, corporations, foundations, and organizations
– ~350k clinical visits / annum
– 4000 DFCI employees between clinical, research and administrative staff
• Centre for Molecular Oncologic Pathology is a DFCI/BWH institute within the division of Medical Oncology.
– CMOP supports collaborative pathology-based research projects that have the potential to result in applications for targeted cancer therapy with a particular focus on prostate cancer
Prepared by Michaela Bowden Ph.D
Prostate Cancer • 238,590 new cases of prostate cancer will be diagnosed in
2013^
• 29,720 deaths are projected for 2013^ – Second-leading cause of cancer death in men
• Higher rate of incidence and mortality in African Americans
• Only established risk factor is AGE (~60% diagnosis in men older than 65 years)
• Standard practice is routine PSA testing for early detection
The major challenge in clinical research of prostate cancer is
distinguishing between indolent and aggressive types with a
tendency to recur. Most research activities in the field focus on
better understanding this distinction.
^ American Cancer Society. Cancer Facts & Figures 2013. Atlanta: American Cancer Society; 2013
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
• Routine preservation method for archiving tumors
• Archived collections are stored for decades and are an invaluable tool for biomedical research
• Large cohorts of samples are required for meaningful clinical discovery
• Extensive RNA fragmentation and cross-linking have been limiting factors for clinical translation applications leading to biomarker discovery
“20 million archival tissue samples are stored annually in US as FFPE blocks but RNA degradation during fixation and storage has prevented their use for transcriptional profiling”
Clin Cancer Res; 18(22) November 15, 2012
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
Diagnosis • 2 specimen collections types
• Fine need aspirate – Less invasive
– Small # cells collected
– H&E and cell block only
• Core biopsy – Large tissue core punch
• Frozen: Difficult to assess morphologically
• Fixed: IHC, stored at RT, no extra cost
– Obligated to archive for 25 year, large accumulation of preserved blocks
• PrCa cannot be diagnosed on frozen sections due to the difficulty in assessing the morphology
Cohort Studies • Globally large scale cohorts
for PrCa have been archived
• In-house (Collaboration with HSPH) – > 3000 PrCa tissue specimens in
biorepository
• Physician Health Study (PHS)
• Physicians Health Follow-up Study (PHFS)
• Swedish Watchful Waiting Cohort (SWWC)
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
• Factors influencing RNA degradation in FFPE tissues – Fixation*
• Thickness of tissue specimen
• Duration of fixation process
• Volume of the formalin solution
– Age
– Water
• Retention of endogenous H2O during tissue processing
• Humid environment resulting in ambient H2O effect
– Temperature
• Effect of humidity is temperature Dependent
– Air
• Oxygenation
* “Unlocking your FFPE archive: Critical factors for molecular analysis of FFPE samples” – Qiagen FFPE Brochure 09/2010
Prepared by Michaela Bowden Ph.D
Project Goals
• To implement an automated method for preparation of RNA-seq libraries from archival FFPE tissues utilizing the TrueSeq kit
– To optimize optimal RNA input requirements
– To establish library size from degraded FFPE
– To establish purification methods for FFPE library clean-up
– To compare manual library preparation and automated method
Prepared by Michaela Bowden Ph.D
Collaboration Workflow
1. Sample Selection
2. RNA Extraction
3. RNA QC and Quantitation
4. Biomek TruSeq Library Preparation
5. Library Purification
6. Manual TruSeq Library Preparation
7. Library Purification
8. RNA Sequencing at Centre for Computational Cancer Biology [CCCB]
9. Sequencing Performance Summary
Prepared by Michaela Bowden Ph.D
Sample Selection
• 3 paired Tumor / Normal FFPE tissue specimens were selected after review by DFCI pathologist – Gleason Score 3+4
• Sample ID’s have been masked to protect patient confidentiality – Prostate Tumor A / Benign A
– Prostate Tumor B / Benign B
– Prostate Tumor C / Benign C
• Tumor or Benign Regions of Interest [ROI] were identified from H&E stains and cores were collected for RNA extraction
Prepared by Michaela Bowden Ph.D
RNA Input
Extraction • Assay: Agencourt®FormaPure® Kit,
Beckman Coulter® [Catalog # A33343]
Quantitation • Assay: Quant-iT™ RiboGreen® RNA
Assay Kit, Invitrogen™ [Catalog # R11490]
• Instrumentation: Perkin-Elmer Victor™ X3
Qualitative Analysis • Assay: Agilent RNA 6000 Nano Kit,
Agilent Technologies [Catalog # 5067‐ 1511]
• Instrumentation: Agilent Bioanalyzer 2100
Tumor A
Benign A
T B
RNA concentrations ranged from 24.4
– 61.1 ng/l, of which 500 ng of total
RNA was utilized for each library as
the input amount.
Prepared by Michaela Bowden Ph.D
TruSeq Method
• The TruSeq RNA method comprises multiple steps
• For adaptation to an automated format, these steps can be broken into three parts that correlate to stop points:
– mRNA purification, fragmentation and cDNA synthesis
– cDNA library construction (end repair, A-tailing and adaptor ligation)
– PCR amplification and product purification
Prepared by Michaela Bowden Ph.D
TruSeq cDNA Library
• cDNA library yields are higher from automated preparations
Prepared by Michaela Bowden Ph.D
SPRIselect Fragment Size Selection
• Initial library had peak max for the amplified cDNA library at approximately 260bp
• Non-specific adaptor amplified peak at 120bp present in all 12 samples.
• An additional fragment >400bp also present in some samples
• SPRIselect – Size selection with magnetic beads
– STEP 1: 0.7X ratio of SPRIselect bead solution to cDNA library volume. Fragments >500bp DNA binds to beads and is discarded.
– STEP 2: The remaining supernatant treated with a 1.1X volume of SPRIselect beads to rebind the DNA fragments between 150bp-400bp.
-10
40
90
140
190
240
32 52 72 92 112 132
Flu
ore
sc
en
ce
Un
its
Time [s]
Benign A -Biomek
Prostate Benign A - PreProstate Benign A - Post
126bp
-10
40
90
140
190
240
290
340
32 52 72 92 112 132
Flu
ore
sc
en
ce
Un
its
Time [s]
Tumor A - Biomek
Prostate Tumor A - PreProstate Tumor A - Post126bp
> 400bp
Low
mol wt
marker:
35bp High mol
wt marker:
10380bp
Prepared by Michaela Bowden Ph.D
Sequencing Performance
• Sequencing was performed on the Illumina HiSeq platform.
• Multiplexed sequencing was performed in such that each lane contained 3 samples.
• 50bp single end reads were mapped using Tophat
• Transcript abundance was calculated using Cufflinks in FPKM units (Fragments per Kilobase of mRNA per 106 reads)
Prepared by Michaela Bowden Ph.D
Biomek v Manual
Prepared by Michaela Bowden Ph.D
Endogenous Controls
• “Normalizers” are commonly employed for quantitative gene expression analysis in clinical tumor samples to correct for cellular differences and RNA quality variance from sample to sample
• Utilizing a panel of 27 housekeeping genes the Biomek and Manual Library preparative methods were compared
y = 1.0951x - 0.8565R² = 0.9765
0
2
4
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12
0 2 4 6 8 10 12
Bio
me
k [L
og2
]
Manual [Log2]
0.0
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4.0
6.0
8.0
10.0
Robot Manual
Log 2
[FK
PM
]GAPDH
0.0
0.5
1.0
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3.0
Robot Manual
Log 2
[FK
PM
]
G6PD
0.0
1.0
2.0
3.0
4.0
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6.0
7.0
Robot Manual
Log 2
[FK
PM
]
OAZ1
Prepared by Michaela Bowden Ph.D
Conclusions
• Generated RNASeq libraries utilizing adapted TruSeq method from 500ng RNA of FFPE origin
• Automated cDNA libraries had a better yield than those prepared manually
• At the transcript level matched Biomek and manual prepped samples showed good concordance
• TruSeq Method is designed for high quality RNA and works extremely robustly for RNA of cell or fresh frozen origin (Routinely used in-house)
• No optimized Illumina protocol is available for FFPE specimens
• Ribosomal RNA removal not an option with TruSeq for FFPE unless large RNA input is utilized (not possible) – rRNA removal highly desirable for
FFPE
Prepared by Michaela Bowden Ph.D
Acknowledgements
• Dana Farber Cancer Institute
– Ashley Rossi [Molecular Technician]
– Svitlana Tyekucheva [Bioinformatics]
– Massimo Loda [CMOP Director]
• Beckman Coulter
– Beena Lee [Senior Scientist]
– Sarah Simons [Field Application Specialist]
