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TACKLING OSTEOARTHRITIS
-Research Tools At Our Disposal
Mahita Kadmiel
July 21, 2005
OSTEOARTHRITIS
• Arthritis -most common medical problem
• No. 1 cause of disability in America.
• arthron = joint itis = inflammation.
arthritis = joint inflammation.
• osteoarthritis, is the most common form of
arthritis
• affects nearly 21 million people in the
United States
MENISCUS
The Meniscus: Shock Absorber for the Knee
Meniscal Tears
• Traumatic tears
From a sudden load being applied to the meniscal tissue which is
severe enough to cause the meniscal cartilage to fail and let go.
Ex. Twisting injury
• Degenerative meniscal tears
Failure of the meniscus over time.
The meniscus becomes less elastic and compliant
May fail with only minimal trauma
Ex. Just getting down into a squat
*Degenerative meniscal tears can lead to osteoarthritis*
Healthy meniscus Torn meniscus
The expression of genetic material in the meniscus
dictates it
Central Dogma of Life
Reverse Transcription
Proteins make a cell what it is
Staining to find
-how the cells
Staining to find
look (anatomy)
-if cells are dead
(TARA)
or alive (viability)
TISSUE (TUMUL)
CELLS
Histology
Cell Biology
Proteins
Biochemistry
Western Blotting
-To detect proteins DNA
ELISA
-To quantify Southern Blotting
-To find copy number
proteins of genes
Northern Blotting
Enzyme Assays RT-PCR Genome Sequencing
-To measure RNA Real-Time PCR
-To study gene
enzyme activity
expression (TUMUL, BASIA
& MYSELF)
Tools used in our lab
RT-PCR
Real-Time PCR
-To study gene
expression
ELISA
-To quantify proteins
Preparation of cDNA or first strand RT
Reverse Transcription
5’ GACCCAAUUGGUCAGCUAAAAAAA 3’ mRNA
5’ GACCCAAUUGGUCAGCUAAAAAAA 3’
Reverse
transcriptase
……TTTTTTT 5’
A, T, G, C
dNTPs Reverse
Transcription
3’ CTGGGTTAACCAGTCGATTTTTTT 5’
1ST strand cDNA (complementary DNA)
PCR : Polymerase Chain Reaction
Reverse Transcriptase-
Polymerase Chain Reaction
-Exponential amplification
End of 35 cycles
236 = millions of copies
PCR products
RT
Marker
RNA
22 23 24
4 copies 8 copies 16 copies 32 copies
Real-Time PCR
SYBR Green Dye
PCR products
Single stranded Double stranded
DNA PCR product
SYBR Green fluoresces brightly only when
bound to double stranded DNA
Ethidium Bromide
Real-Time PCR
Quantitative method
Most reliable for mRNA (gene) expression
Small amounts of RNA required / tissue
Amplification monitored by fluorescence in real-time
96 well plate
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2 Theoretical and Ideal
2 4
10000000000
3 8 1000000000
4 16
AMOUNT OF DNA
100000000
5 32 10000000
1000000
6 64 100000
7 128 10000
8 256 1000
100
9 512
10
10 1,024 1
11 2,048 0 5 10 15 20 25 30 35
12 4,096 PCR CYCLE NUMBER
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288 Practical !!!!!
20 1,048,576
1600000000
21 2,097,152
1400000000
22 4,194,304
AMOUNT OF DNA
1200000000
23 8,388,608
1000000000
24 16,777,216
800000000
25 33,554,432
600000000
26 67,108,864
400000000
27 134,217,728
200000000
28 268,435,456
0
29 536,870,912
0 5 10 15 20 25 30 35
30 1,073,741,824
PCR CYCLE NUMBER
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
SERIES OF 10-FOLD DILUTIONS
Standard curve generated using serial dilution
Melting / Dissociation Curve
primer dimer
E Enzyme Protein molecule that performs a
chemical reaction
L Linked
Linking an enzyme to an
assay/test
I Immuno
S
Attachment of antibodies
Sorbent
A Assay
Test to find out something
Technique based on antigen-antibody reaction
Examples: HIV tests &PGE2
Well with antibodies Well with antibodies and BSA added
Well with antibodies,
antigen binding sites BSA, and test sample
Well in a microtiter plate
Antibody structure
Well after washes with
wash buffer
Well after adding substrate
Color developed due to the
formation of a substrate
Secondary antibody linked to
an enzynme is added to the well
Well after removing
excess antibody
Other Techniques
• Genome Sequencing
– To find out
» the base composition (A, T, G, C)
» The order in which the bases are arranged
• Northern Blotting ( mRNA expression)
• Southern Blotting (copy number)
• Western Blotting ( protein expression)
Southern / Northern Blotting
Western Blotting
IL-1 RT-PCR IL-1
TNF iNOS
COX-2
MMP
ELISA PGE2
iNOS
Colorimetric Assays
GAG
Nitrate &
Nitrite
NO
MMP COX-2
PGE2
(GAG)
Degrades tissue
Questions???????
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