Transcript Molecular biology Tools

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
look (anatomy)
(TARA)
TISSUE
CELLS
Staining to find
-if cells are dead
or alive (viability)
(TUMUL)
Histology
Cell Biology
Proteins
Biochemistry
Western Blotting
-To detect proteins
DNA
ELISA
-To quantify
proteins
Enzyme Assays
-To measure
enzyme activity
Southern Blotting
-To find copy number
of genes
RNA
Northern Blotting
RT-PCR
Real-Time PCR
-To study gene
expression (TUMUL, BASIA
& MYSELF)
Genome Sequencing
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’
5’ GACCCAAUUGGUCAGCUAAAAAAA 3’
Reverse
transcriptase
……TTTTTTT 5’
A, T, G, C
dNTPs
Reverse
Transcription
3’ CTGGGTTAACCAGTCGATTTTTTT 5’
1ST strand cDNA (complementary DNA)
mRNA
PCR : Polymerase Chain Reaction
Reverse TranscriptasePolymerase Chain Reaction
-Exponential amplification
End of 35 cycles
236 = millions of copies
PCR products
RT
Marker
RNA
22
4 copies
23
8 copies
24
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
AMOUNT OF DNA
Theoretical and Ideal
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
5
10
15
20
25
30
35
PCR CYCLE NUMBER
Practical !!!!!
1600000000
AMOUNT OF DNA
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
1400000000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
5
10
15
20
25
PCR CYCLE NUMBER
30
35
SERIES OF 10-FOLD DILUTIONS
 Standard curve generated using serial dilution
Melting / Dissociation Curve
primer dimer
E
L
Enzyme
Linked
I
S
Sorbent
A
Assay
Immuno
Protein molecule that performs a
chemical reaction
Linking an enzyme to an
assay/test
Attachment of antibodies
Test to find out something
 Technique based on antigen-antibody reaction
 Examples: HIV tests &PGE2
Well with antibodies Well with antibodies and BSA added
antigen binding sites
Well with antibodies,
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
TNF
iNOS
RT-PCR
ELISA
Colorimetric Assays
NO
MMP
Degrades tissue
COX-2
PGE2
(GAG)
IL-1
iNOS
COX-2
MMP
PGE2
GAG
Nitrate &
Nitrite
Questions???????