After MAEC culture with morpholino oligomer, total RNA was purified with an RNeasy mini kit (Qiagen, Germantown, MD), followed by DNaseI treatment (Sigma-Aldrich, St. Louis, MO). cDNA was prepared with an RT Omniscript kit (Qiagen) using oligo-dT. Real-time polymerase chain reaction (PCR) was carried out with SYBR green PCR (Qiagen) using previously described primers and PCR reaction conditions.12 (link),13 (link) Real-time data were analyzed with the ΔΔCt method. sFLT protein quantification from the conditioned culture medium and mouse serum was performed by Mouse sVEGF R1/Flt-1 ELISA (R&D Systems, Inc., Minneapolis, MN) following the manufacture's instruction.
Protocol detail
Quantifying sFLT Protein Expression
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Cdna
Conditioned culture medium
Elisa
Flt 1
Morpholino
Mouse
Oligo dt
Polymerase chain reaction
Primers
Protein
Reaction conditions
Real time polymerase chain reaction
Serum
Sybr green
Corresponding Organization : University of Utah
Other organizations : Fourth Affiliated Hospital of China Medical University, China Medical University
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Variable analysis
independent variables
- MAEC culture with morpholino oligomer
- Total RNA expression
- SFLT protein quantification from the conditioned culture medium and mouse serum
- DNaseI treatment (Sigma-Aldrich, St. Louis, MO)
- CDNA preparation with an RT Omniscript kit (Qiagen) using oligo-dT
- Real-time polymerase chain reaction (PCR) with SYBR green PCR (Qiagen) using previously described primers and PCR reaction conditions
- Positive control: Not specified
- Negative control: Not specified
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