BiOstic™ FFPE Tissue DNA
Isolation Kit (cat# 12250-50)
For use with paraffin embedded tissues
and
BiOstic™ Paraffin Removal Reagent
(cat# 12251-50)
WWW.MOBIO.COM
Overview
• Marketing Overview
• R&D Data Review
• Sales Tools
What FFPE stands for?
•
A formaline fixed paraffin embedded tissue (FFPE
Tissue) is a piece of tissue which is first separated from
a specimen animal by dissection or biopsy. Then, this
tissue is fixed in order to prevent it from decaying or
degeneration and to examine it clearly under a
microscope for histological, pathological or cytological
studies. This fixed tissue is then embedded in the wax to
be cut into fine sections and stain with Hemotoxylin and
Eosin Stain. After that microtoming is done, the tissue
can be studied under a microscope.
•
Fixation is the process by which the tissue is
immobilized, killed and preserved for the purpose of
staining and viewing it under a microscope. Fixation
makes tissue permeable to staining reagents and crosslinks its macromolecules so that they are stabilized and
locked in position. Many fixatives are used for this
purpose for example, Bouine solution, Formaline, or
Liquid Nitrogen.
What is the BiOstic™ FFPE Tissue
DNA Isolation Kit?
• A way to extract genomic DNA from formalin fixed
paraffin embedded (FFPE) tissues.
– used in cancer studies to view the morphology of potentially
cancerous cells.
– Genomic DNA used in SNP (Single nucleotide
polymorphisms) genotyping, HLA (Human Leucocyte
Antigen: Molecules that can identify cells as self providing the
immune system to attack itself) and other genetic analysis.
• Why is it different?
– uses a novel method to directly melt the wax away from the
tissue
– Does not use toxic solvents such as xylene to deparaffinize
the tissue.
– Yields are higher and DNA is more pure = lower Ct values
and more sensitive detection.
BiOstic™ Paraffin Removal Reagent
• BiOstic™ Paraffin Removal Reagent
is a non-hazardous, non-toxic
alternative to xylene
– For those who still prefer to de-wax using
solvents
– Can be used with the BiOstic™ FFPE
Tissue DNA Isolation Kit
– Cat # 12251-50
Features and Benefits
• Higher yields of DNA- less protocol steps
results in less chances for loss of sample
and higher yields of DNA
• More sensitive results- higher purity due to
non-toxicity provides lower Ct values in realtime PCR.
• Safe and non-toxic- no xylene or solvents
required
Note: Ct value= Concentration of DNA molecules (in moles)
multiplied by time.
Customer Overview
Who are the customers?
• Cancer research labs
– Breast cancer labs
• HLA (Human Leukocyte
Antigen) Labs
• Clinical research labs
How do I find them? What
do they buy now?
• Histology supplies,
microscope slides, tissue
stains,
Where do they work?
• Universities
• Pathology Labs
• Cancer institutes
• Large pharmaceutical
companies
Why do they need this
kit?
• Higher DNA recovery without
toxic solvents
• Laboratory safety
• No PCR inhibition
• Complete kit – no extra
reagents needed.
Competitor Overview
Competition Spec
Qiagen
56404
MO BIO
12250-50
160 minutes
140 minutes
Toxic Solvent free
No
YES
Buffers ready to use?
No
YES
$179
$139
$3.58/prep
$2.78/prep
Speed
Cost (US)
Price per prep (US)
R&D Data
Protocol
Key steps:
Digestion- tissues are melted directly in a high powered buffer
that optimized proteinase K activity.
900C incubation is critical for removing cross-links in DNA that
inhibit PCR.
Purification- DNA is isolated quickly on Spin Filters and without
inhibitors.
FFPE tissue purified DNA is typically
low molecular weight
L
MB
MB
Q
Q
1 kb
500bp
DNA prepared with the MO BIO Kit shows higher molecular weight
distribution as compared to the Qiagen protocol using xylene.
High molecular weight DNA purified with
the BiOstic™ FFPE Tissue DNA Kit
• There are two protocols available for
the BiOstic FFPE Tissue kit:
- Option 1: direct melting of the paraffin
- Option 2: use of the non-toxic solvent BiOstic
paraffin removal reagent
- Both yield similar results but there is a
difference in timing and personal
preference.
High molecular weight DNA purified with
the BiOstic™ FFPE Tissue DNA Kit
L
4 kb
PR PR M
M
PR = Paraffin
removal protocol
M = Melt protocol
1 kb
Isolated DNA has high molecular weight species when
separated with a 1.5% agarose gel
MO BIO outperformed Qiagen in qPCR
results for DNA isolation from FFPE tissue
DNA isolated from the BiOstic™ FFPE Tissue DNA
Isolation Kit is ready for qPCR
1 CT earlier detection = 2 fold increase in sensitivity
FAQs- Technical Questions
•
How many mgs of FFPE tissue should I use?
– We tested up to 15 mg of tissue. As little as 1 slice may be used or
2-3 slices can be combined to increase yields.
•
What factors impact the yields?
– The type of tissue, length of time it was in fixative, how even the
slices were made, how much tissue is in the slice, and how much
sample you use.
•
What if I want to concentrate the eluted DNA?
– If you are using only 1 slice and expect yields to be in the 10ng/µl
range, depending on your application, concentration may be helpful.
Elute the sample in 100 µl to maximize the full recovery off of the
membrane and then use standard ethanol precipitation techniques
(ask technical service for our protocol). Alternatively, the UltraClean
15 Kit may be used to concentrate the sample down to a volume of
12 µl.
FAQs- Technical Questions
• What are expected yields?
– 1-3 µg from 3 sections of 5 microns is average.
Measuring yield on a Nanodrop and using
Picogreen will give different results depending
on the amount of ssDNA present in the sample.
Nanodrop will detect both ssand dsDNA and
Picogreen will detect only dsDNA.
– Using the MO BIO Kit with 12 mg of tissue, we
have seen yields of up to 8 µg using a
Nanodrop.
Sales Tools
Spin Selling your customer – What to ask
Situation, Problem, implications, need/payoffs
• S: Are you isolating DNA from FFPE
Tissue?
• S: Are you currently using solvents such as
xylene to deparaffinize the tissue?
• P: Does the protocol you are currently using
has too many steps, and thus, affects the
amount of yield ?
• P: Does the method with solvents affect the
DNA purity?
SPIN Selling your customer – What to ask
Situation, Problem, Implications, Need/payoffs
• I: The solvent wax removal method usually takes
longer. How does that affect your ability to get work
done?
• I: The use of toxic solvents may affect the quality of
the DNA yield and lab safety. How does that affect
your research?
• N: If you could use a product that would provide a
safer, faster and easier way to isolate DNA from
FFPE tissue, would you buy it?
• N: If you could use a product that would provide a
gentle method to isolate DNA from FFPE tissue,
and thus have more sensitive results and lower Ct
values in real-time PCR due to higher DNA purity
(contaminant-free), would you buy it?
Sales Tools / Promotional Activities
• Sales Summary and Competitive Analysis
• BiOstic™ FFPE Tissue DNA Isolation Kit sample:
12250-S
• BiOstic™ literature: MC 101
• BiOstic™ pull up banner
• Website
• Press Release in January 2009
• Stickers being placed in the UltraClean Tissue and
Cells DNA Isolation Kits
• Promotion at conferences: Tri-conference, Feb
2009; AACR, April 2009; and eventually AMP 2009,
October 2009.
Ordering information and Contacts
• Cat#12250-50 (50 preps) and 12250-S (sample
size)
• R&D: Suzanne kennedy, skennedy@mobio.com
• Technical Support: Nik von Atzigen,
nvonatzigen@mobio.com
• Marketing- Peggy Maunaury,
pmaunaury@mobio.com