research final ppt

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


2/19

LIMITED

CAPACITY

FOREARLY

DIAGNOSIS


3/19

0

20

40

60

80

100

STAGE 4

5-YEAR SURVIVAL RATE


4/19

0

20

40

60

80

100

STAGE 1


50%


5/19

0

20

40

60

80

100

STAGE 4


2%


6/19

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


7/19

cancercellsexpress

markersspecific to the

tissue oforigin

E ELLS


8/19

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


9/19

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


10/19

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


11/19

pre-experime talstudy

intending to provide

baseline knowledge toassist further biomarker

research


12/19

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


13/19

RESULTS AND

discussion


14/19

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.


15/19


16/19


R

ES

U

L

T

S


17/19


R

ES

U

L

T

S


18/19

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


19/19

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

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