TITLE: Ultrasound-Mediated Vascular Gene Transfection by Cavitation of EndothelialTargeted Cationic Microbubbles
AUTHORS: Aris Xie, BS, Todd Belcik, RDCS, Yue Qi, MD, Terry K. Morgan, MD, PhD,
Shivam A. Champaneri, MD, Sarah Taylor, PhD, Brian P. Davidson, MD, Yan Zhao, MS,
Alexander L. Klibanov, PhD, Michael Kuliszewski, BS, Howard Leong-Poi, MD, Azzdine
Ammi, PhD, Jonathan R. Lindner, MD
APPENDIX
Supplement – Methods: rtPCR
Total RNA was isolated from muscle tissue from mice receiving either P-selectin-targeted or
non-targeted plasmid-coupled microbubbles (n=18 each group) exposed to an MI of 0.6 or 2.4.
Muscle was homogenization in Trizol reagent (Invitrogen) followed by chloroform extraction
and ethanol precipitation/wash according to the manufacturer’s instructions. This lysate was then
purified with a Bio-Rad Aurum Total RNA Mini Kit (Bio-Rad, Hercules, CA). Real time PCR
was performed using iScriptTM cDNA Synthesis Kit and SYBR Green (BIO-RAD, Hercules, CA,
USA) following manufacturer’s protocols. Experimental cDNA samples were run in triplicate
with quantities calculated from standard curves generated from serial dilutions of cDNA from a
control sample. Primer sequences used to amplify transcripts were: GAPDH (Glyceraldehyde 3phosphate dehydrogenase) (forward: 5’- TTG CCA TCA ACG ACC CCT TC -3’ and reverse:
5’- TTG TCA TGG ATG ACC TTG GC -3’) ; Luciferase (forward: 5’- CTA GCA AAA TAG
GCT GTC CC -3’ and reverse: 5’- GAC GAT AGT CAT GCC CCG CG-3’. Expression of
cDNA was normalized to GAPDH levels to control for variation in reaction inputs.
2
Supplemental Figure
3
Acoustic Response (V)
2.5
2
With Bubbles 1 MPa
1.5
No Bubbles 1 MPa
with Bubbles 0.5 MPa
1
No Bubbles 0.5 MPa
0.5
0
1 2 3 4 5 6 7 8 9 10111213141516171819202122232425
Pulse number
Mean (±SD) peak-to-peak amplitude of microbubble response (peak-to-peak) measured by a
passive cavitation detector for solutions containing either cationic microbubbles without cDNA
or no microbubbles during sequential 1 MHz ultrasound pulses at 0.5 and 1.0 MPa peak negative
acoustic pressure.