Computational protocol: RNA-Eluting Surfaces for the Modulation of Gene Expression as A Novel Stent Concept

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Protocol publication

[…] First of all, total RNA from PLGA/RNA transfected EA.hy926 and hVECs was isolated using Aurum™ total RNA mini kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNA was quantified and 200 ng of each RNA sample was utilized for the iScript™ cDNA Synthesis Kit from (Bio-Rad) according to the manufacturer’s instructions for reverse transcription. Primer design was done with NCBI primer-blast, the primer sequences in , synthesized by Operon (Köln, Germany), were used for qRT-PCR. Poly (IC) dsRNA from R&D systems (Minneapolis, MN, USA) served as positive control for immune stimulation. PCR mixes contained IQ™SYBR®Green Supermix (Bio-Rad), 400 nM forward and reverse primer, and 2 ng of cDNA in a total volume of 15 µL. All samples were performed in triplicates. Normalized gene expression was calculated by the threshold cycle (ΔCt) method using GAPDH as a reference. [...] This technique which was established to detect siRNA-mediated mRNA cleavage was first described by Soutschek et al. in 2004 []. Later Davis et al. used this technique to verify siRNA-mediated mRNA cleavage in a human phase I clinical trial []. This technique detects cleaved mRNA and is especially suitable to confirm the mRNA degradation dependent on the base pair sequence of the siRNA in transfection assays. Therefore, 50 µg of PLGA and 3 µg ICAM-siRNA were dried on glass slides, the glass slides were laid down on confluent EA.hy926 cells for 48 h and cells were stimulated for 12 h with the above stated TNF-α concentration. Subsequently, the RNA was isolated as described above. For our study we used components of the commercially available First Choice® RLM-RACE Kit from Life technologies (Darmstadt, Germany). The kit is delivered with RNA-Adapter and T4 RNA Ligase to conduct the 5′ RACE Adapter Ligation. We used 2 µg of isolated RNA for ligation. Also the kit contains M-MLV Reverse Transcriptase for first strand cDNA synthesis where we used 200 ng of ligated RNA. The user has to design gene-specific primers for the first strand synthesis and also for the PCR of the Outer and Inner PCR which are following after first strand cDNA synthesis. Primer design was done again with the software ‘Primer3’ [] and Primer Premier 5 (PREMIER Biosoft International). Primer sequences that were used for 5-RLM-RACE-PCR are shown in . All PCR reactions (Outer and Inner PCR) were conducted as a qRT-PCR and contained IQTMSYBR®Green Supermix from Bio-Rad (Hercules, CA, USA), 400 nM forward and reverse primer and 2 ng of cDNA in a total volume of 15 µL. All PCR reactions were performed in triplicates. For better understanding of this technique we also designed an illustration (). […]

Pipeline specifications

Software tools Primer-BLAST, Primer3, Primer Premier
Application qPCR
Organisms Homo sapiens
Diseases Thrombosis, Coronary Restenosis, Atherosclerosis