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8/9/2019 Research Final PPT
1/19
Abalos KS, Abarquez PD, Achero IP, Agdamag AC, Aguro MD,
Alacapa JV, Alfabeto AB, Aligam KG, Almanon CM, Almeda TC,
Alonte JD, Angeles RB, Araneta KS, Arce BP, Asunto PQ, Avila CS
INVESTIGATORS
Francisco S. Chung Jr., PhD
ADVISER
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LIMITED
CAPACITY
FOREARLY
DIAGNOSIS
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0
20
40
60
80
100
STAGE 4
5-YEAR SURVIVAL RATE
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0
20
40
60
80
100
STAGE 1
5-YEAR SURVIVAL RATE
50%
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0
20
40
60
80
100
STAGE 4
5-YEAR SURVIVAL RATE
2%
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POOR
SURVIVAL
RATES
THE PROBLEM OF LUNG CANCER
NEE FORNEE FOREARLEARL
IAGNOSISIAGNOSIS
UNAVAILABILITUNAVAILABILIT
OF APPROVEOF APPROVE
SENSITIVESENSITIVE
IAGNOSTICIAGNOSTIC
TOOLSTOOLS
A SENSITIVE AN EFFICIENT
SOLUTION IN EVELOPMENT
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cancercellsexpress
markersspecific to the
tissue oforigin
E ELLS
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studies exploring the use of
l gca cer biomarkersin
the Philippines are lacki g
Nomarkersfor lung cancer
yet for Fi l i i o patients
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MAIN OBJECTIVE
To develop the o timizedco ditio s
for SYBR Green I-based one-step qRT-
PCR for the simultaneousdetection of PLUNC, CEACAM5
and KRT-19 biomarkers in
paraffi ized lung parenchyma tissueof Filipino patients
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SPECIFIC
OBJECTIVESTo determineTo determine
1.1. if ifRNARNA can be extracted fromcan be extracted from
paraffinizedparaffinized lung parenchyma tissuelung parenchyma tissue2. the optim mco ditio s for SYBR
Green I-based one-step qRT-PCR for
detection of selected biomarkers3. determine which among previously
studied biomarkers have a pote tial
use in the Philippines
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pre-experime talstudy
intending to provide
baseline knowledge toassist further biomarker
research
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pre-experime tal
(+) NSCLC samples obtained from the UP-PGHDepartment of Laboratories
Co v e ie cesampli g strict release of samples byhospital
samples - randomly subjected to qRT-PCR and/ortroubleshooting methods in different group combinations
Primers chosen based on study by Cheng et al. (showprimers) and several other supporting studies best biomarker candidates in the literature
use ofACTB as a housekeeping gene housekeeping gene is present in all tissue types
provides a reference value against which the amplification of
each biomarker can be compared to
STUDY
DESIGN
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RESULTS AND
discussion
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OLIGONUCLEOTIDE DESIGN
R
E
S
U
L
TS
BLAST ANALYSIS
primers could anneal to sequences
other than what it is designed for
- producing nonspecific products
primers may amplify a certain double-
stranded product- not necessarily the desired amplicon
Necessary to visualize PCR
products through gelelectrophoresis to determine if
the amplified products have the
desired amplicon size.
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OLIGONUCLEOTIDE DESIGN
R
ES
U
L
T
S
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OLIGONUCLEOTIDE DESIGN
R
ES
U
L
T
S
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acceptable or near-optimal quantitation curves for qRT-
PCR for ACTB and KRT19.
ACTB sequence - housekeeping gene in future
biomarker studies KRT19 - also suggested as valid biomarker for lung
cancer detection
PLUNC and CEACAM5 may not be applicable for use
with Filipino patients; may have a faulty design. RNA can be extracted from paraffinized lung tissue
better amplification results from a positive control
suggest the possibility of loss of mRNA integrity
CONCLUSION
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replicate study using:
1. betterprimerswith more ideal designs
2. templates or samples that have moreguaranteed mRNA integrity
fresh samples may be opted but difficult to obtain
more recently paraffanized tissues have less
likelihood of RNA degradation
Paraffinized blocks 6 mos. old or less may be
more ideal to use
RECOMMENDATION
S