The SPARC small interfering RNA (siRNA) sequences were designed with the help of a siRNA designer programme (Imgenex, Sorrento Valley, CA, USA).
A mix of two different dsRNAs was used: BRCA1-A (ID 5479, Ambion) mapped to exon 12 and BRCA1-B, designed with Cenix Designed siRNAs program (Ambion, www.ambion.com/siRNA), mapped to exon 24.
Targets for each rat EphA5, EphB1 or EphB2 siRNA were designed with reference to the NCBI library, and the uniqueness of sequence was ascertained with the NCBI nucleotide BLAST program.
Although the original pInv-H1 construct was made with the siRNA sequence targeting the mRNA of DC-SIGN, a C-type lectin receptor highly expressed on dendritic cells and involved in binding to ICAM-2 [20], ICAM-3 [21], and HIV-1 gp120 [23], [23], the oligonucleotides were designed with EcoRI and BglII sites flanking the siRNA sequence, allowing for the convenient exchange of siRNA sequences.
These siRNAs are designed with unequal stabilities of the 3' and 5' bases, to reduce the risk of off-target effects.
When the goal is to block the expression of genes with molecules of siRNA (small interfering RNA), the synthetic DNA can be designed with a section complementary to the RNA so that the RNA will loosely attach to it.
SiRNA-mediated knockdown was performed using On-Targetplus SMARTpool siRNA mixes (Thermo Scientific), designed with dual-strand modification that decreases off target effects and containing a mix of four individual siRNAs Table 5).
While this may be essential for host cell viability, it is not clear that it is essential for expression of retroviral genes, thus siRNA screens designed with tat-mediated expression of a reporter gene may not provide a reliable readout for factors involved in post-integration DNA repair.
The experimental results showed that siRNA-G41 successfully silenced PhaTF106, PhaTF157 and PhaTF187, demonstrating that it is feasible to simultaneously silence multiple genes with a carefully designed siRNA and that siRNA can silence the target gene even with 4 mismatches to its target genes.
Three stealth siRNA targeted to the ZNF703 mRNA (Invitrogen) were designed along with the corresponding stealth siRNA control.
