Phenotype of Plaque

UW Cardiovascular Pathology Training Program
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Phenotype of Plaque versus Medial Human Carotid Smooth Muscle Cells

Summary of Experiments Graphs

Exp 1 total RNA 9/20/01
UTA 1	# of Filters	Phosphoimager Exposures
10 million cpm per filter	2	3
20 million cpm per filter	2	3
40 million cpm per filter	2	3
80 million cpm per filter	2	3

Exp 2 total RNA 9/27/01
UTA 2
10 million cpm per filter	2	3
20 million cpm per filter	2	3
40 million cpm per filter	2	3
48 million cpm per filter	2	3
Exp 3 amplified total RNA 12/6/01
ARNA 1
1 ug starting RNA 170 million	2	3
1 ug starting RNA 100million	2	3
0.1 ug starting RNA 50 million	2	3
0.1 ug starting RNA 25 million	2	0* maximun intensity too low to import

Exp 4 amplified total RNA 12/20/01
ARNA 2
1 ug starting RNA 250 million	2	4
1 ug starting RNA 250million	2	4
0.1 ug starting RNA 250 million	2	4
0.1 ug starting RNA 200 million	2	4

Exp 5 total RNA 3/11/02
38 million cpm per filter	21	2

Experiments to establish the protocol for hybridization to Research Genetics filters. We used Stratagene Universal Target total RNA to make P33 labeled probe according to the manufacturer. Experiments 1 and 2 explored the amount of first strand cDNA probe required for hybridization. Experiments 3 and 4 used Arcturus T7 RNA amplification to generate an aRNA target that was labeled with P33 using random 9mers. Experiment # 5 used 20 filters from the same lot of filters that had been hybridized and stripped on four previous occasions. Experiments #1-4 were done on Research Genetics GF211 filters lot 000223E - filters11, 12,13,15,16,17,18,19. Experiment #5 was done on lot 000223E - filters 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 69, 72, 73, 74, 75, 76, 77.

Exp 1 total RNA 9/20/01

UTA 1

Exp 2 total RNA 9/27/01

UTA 2

Exp 3 amplified total RNA 12/6/01

ARNA 1

ARNA 2

Exp 5 total RNA 3/11/02