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Reliable Procedure to Maintain RNA Quality During Laser Capture Microdissection of Human Aortic Smooth Muscle Cells
Abstracts CSANZ 2012 Abstracts
S271
652
653
Rate of Aneurysm Formation is Reduced with Increased Aortic Stiffness
Reliable Procedure to Maintain RNA Quality During Laser Capture Microdissection of Human Aortic Smooth Muscle Cells
R. Jeremy 1,∗ , K. Vis 2 , A. DeWit 2
C. Woo 1,∗ , C. Kong 1,∗ , Y. Chan 3 , C. Wong 3 , J. Leong 2 , C. Lee 1 , V. Sorokin 2
1 University
of Sydney, Australia 2 Erasmus University, Netherlands Increased aortic stiffness occurs in Marfan syndrome and other thoracic aortopathies but whether changes in stiffness alter rate of aneurysm growth is unknown. Aortic stiffness index (SI) was calculated from simultaneous measurements of aortic root geometry (2D echo) and brachial blood pressure in 149 patients (55 Marfan, 47 bicuspid aortic valve and 47 isolated familial aortopathy). Change in maximal aortic diameter (annual echo) during follow-up was calculated as mm/year. Patients were grouped by tercile of aortic stiffness. Demographics and rate of change in aortic size were compared between groups by ANOVA (SPSS 19). Group n Age (years) Follow-up (years) BP systole (mmHg) BP diastole (mmHg) Aorta max (mm) Aortic index (mm m−2 ) Aortic change (mm/yr) Stiffness index
SI < 8
SI = 8–16
45 34.5 ± 12.2 8.2 ± 6.9 113.8 ± 12.6 71.6 ± 8.9 40.8 ± 5.6 21.7 ± 4.4 0.68 ± 0.39 5.3 ± 1.7
49 41.5 ± 15.8 7.7 ± 6.5 121.6 ± 10.0† 74.5 ± 7.0 43.3 ± 5.5 21.9 ± 3.6 0.49 ± 0.40 11.6 ± 2.2‡
SI > 16 55 49.6 ± 16.1‡ , ¤ 7.4 ± 6.5 126.6 ± 13.8‡ 76.2 ± 8.2* 44.4 ± 5.3† 22.8 ± 3.2 0.25 ± 0.30‡,§ 29.5 ± 10.9‡,§
Mean ± SD. ∗
p < 0.02 vs SI < 8.
†
p < 0.01 vs SI < 8.
‡
p < 0.001 vs SI < 8. ¤ p < 0.02 vs SI 8–16.
§
p < 0.001 vs SI 8–16.
Greater aortic stiffness is associated with higher systolic blood pressure but lesser rates of increase in aortic aneurysm size, suggesting prognostic utility of aortic stiffness measurements. http://dx.doi.org/10.1016/j.hlc.2012.05.663
1 Department
of Surgery, Yong Loo Lin School of Medicine, National University of Singapore 3 Cardiovascular Research Institute, National University Heart Centre, Singapore, National University Health System, Singapore 2 Department of Cardiac, Thoracic & Vascular Surgery, National University Heart Centre, Singapore, National University Health System, Singapore 3 Department of Biopolis Shared Facilities, Genome Institute of Singapore Background: Study of the characteristic of aortic smooth muscle cells (SMC) is crucial for understanding underlying mechanisms that lead to atherosclerotic disease. Hence, we have developed a reliable protocol, which involves the extraction of micoRNA and messenger RNA (mRNA) from SMC of fresh aorta using laser capture microdissection (LCM). Methods: Aortic specimens were collected in the operation theatre during coronary artery bypass surgery. Attention was paid to shorten the duration of tissue dissection to processing. Fresh frozen aortic specimens were embedded in the Optimal Cutting Temperature Compound (OCT). The sections of 10 m thick were stained with Arcturus Histogene Frozen Section Staining Kit (Applied Biosystems). LCM was carried out on LMPC technology in MicroBeam system (Carl Zeiss) with sections placed on Polyethylene Naphthalate Membrane (PEN) slides. We managed to obtain 400ng of total RNA with satisfactory quality. The extracted total RNA were amplified and labeled with NUGEN WT-OVATION FFPE RNA and NUGEN ENCORE BIOTIN labeling kit respectively. The labeled RNA was hybridised onto the Affymetrix U133 GeneChip Arrays. Results: With 50ng of total RNA, amplification yielded >9 g of cDNA for downstream mRNA profiling with the Affymetrix U133 plus 2 array. The QC results of the array proved that the amplified RNA is of sufficient good quality for mRNA profiling. Conclusion: This method of extraction is able to yield a good amount of reasonable quality RNA from laser dissected SMC, it serves as a useful platform for mRNA and miRNA profiling of SMC to better understand the mechanisms of atherosclerotic process. http://dx.doi.org/10.1016/j.hlc.2012.05.664
ABSTRACTS
Heart, Lung and Circulation 2012;21:S143–S316
S271
652
653
Rate of Aneurysm Formation is Reduced with Increased Aortic Stiffness
Reliable Procedure to Maintain RNA Quality During Laser Capture Microdissection of Human Aortic Smooth Muscle Cells
R. Jeremy 1,∗ , K. Vis 2 , A. DeWit 2
C. Woo 1,∗ , C. Kong 1,∗ , Y. Chan 3 , C. Wong 3 , J. Leong 2 , C. Lee 1 , V. Sorokin 2
1 University
of Sydney, Australia 2 Erasmus University, Netherlands Increased aortic stiffness occurs in Marfan syndrome and other thoracic aortopathies but whether changes in stiffness alter rate of aneurysm growth is unknown. Aortic stiffness index (SI) was calculated from simultaneous measurements of aortic root geometry (2D echo) and brachial blood pressure in 149 patients (55 Marfan, 47 bicuspid aortic valve and 47 isolated familial aortopathy). Change in maximal aortic diameter (annual echo) during follow-up was calculated as mm/year. Patients were grouped by tercile of aortic stiffness. Demographics and rate of change in aortic size were compared between groups by ANOVA (SPSS 19). Group n Age (years) Follow-up (years) BP systole (mmHg) BP diastole (mmHg) Aorta max (mm) Aortic index (mm m−2 ) Aortic change (mm/yr) Stiffness index
SI < 8
SI = 8–16
45 34.5 ± 12.2 8.2 ± 6.9 113.8 ± 12.6 71.6 ± 8.9 40.8 ± 5.6 21.7 ± 4.4 0.68 ± 0.39 5.3 ± 1.7
49 41.5 ± 15.8 7.7 ± 6.5 121.6 ± 10.0† 74.5 ± 7.0 43.3 ± 5.5 21.9 ± 3.6 0.49 ± 0.40 11.6 ± 2.2‡
SI > 16 55 49.6 ± 16.1‡ , ¤ 7.4 ± 6.5 126.6 ± 13.8‡ 76.2 ± 8.2* 44.4 ± 5.3† 22.8 ± 3.2 0.25 ± 0.30‡,§ 29.5 ± 10.9‡,§
Mean ± SD. ∗
p < 0.02 vs SI < 8.
†
p < 0.01 vs SI < 8.
‡
p < 0.001 vs SI < 8. ¤ p < 0.02 vs SI 8–16.
§
p < 0.001 vs SI 8–16.
Greater aortic stiffness is associated with higher systolic blood pressure but lesser rates of increase in aortic aneurysm size, suggesting prognostic utility of aortic stiffness measurements. http://dx.doi.org/10.1016/j.hlc.2012.05.663
1 Department
of Surgery, Yong Loo Lin School of Medicine, National University of Singapore 3 Cardiovascular Research Institute, National University Heart Centre, Singapore, National University Health System, Singapore 2 Department of Cardiac, Thoracic & Vascular Surgery, National University Heart Centre, Singapore, National University Health System, Singapore 3 Department of Biopolis Shared Facilities, Genome Institute of Singapore Background: Study of the characteristic of aortic smooth muscle cells (SMC) is crucial for understanding underlying mechanisms that lead to atherosclerotic disease. Hence, we have developed a reliable protocol, which involves the extraction of micoRNA and messenger RNA (mRNA) from SMC of fresh aorta using laser capture microdissection (LCM). Methods: Aortic specimens were collected in the operation theatre during coronary artery bypass surgery. Attention was paid to shorten the duration of tissue dissection to processing. Fresh frozen aortic specimens were embedded in the Optimal Cutting Temperature Compound (OCT). The sections of 10 m thick were stained with Arcturus Histogene Frozen Section Staining Kit (Applied Biosystems). LCM was carried out on LMPC technology in MicroBeam system (Carl Zeiss) with sections placed on Polyethylene Naphthalate Membrane (PEN) slides. We managed to obtain 400ng of total RNA with satisfactory quality. The extracted total RNA were amplified and labeled with NUGEN WT-OVATION FFPE RNA and NUGEN ENCORE BIOTIN labeling kit respectively. The labeled RNA was hybridised onto the Affymetrix U133 GeneChip Arrays. Results: With 50ng of total RNA, amplification yielded >9 g of cDNA for downstream mRNA profiling with the Affymetrix U133 plus 2 array. The QC results of the array proved that the amplified RNA is of sufficient good quality for mRNA profiling. Conclusion: This method of extraction is able to yield a good amount of reasonable quality RNA from laser dissected SMC, it serves as a useful platform for mRNA and miRNA profiling of SMC to better understand the mechanisms of atherosclerotic process. http://dx.doi.org/10.1016/j.hlc.2012.05.664
ABSTRACTS
Heart, Lung and Circulation 2012;21:S143–S316