Lipofectamine® RNAiMAX Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA sequence:sequences are 5′-CAUCUCCUAUGGCGGUGGU (siNB1i) and 5′-UGGUCCUCCUCAAGGCAGU (siNB2i) Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 3 days at 37°C.

Protocol tips
siRNA sequence:sequences are 5′-CAUCUCCUAUGGCGGUGGU (siNB1i) and 5′-UGGUCCUCCUCAAGGCAGU (siNB2i) Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 3 days at 37°C.

Publication protocol

Two different siRNAs targeting Nullbasic were synthesized. The sequences are 5′-CAUCUCCUAUGGCGGUGGU (siNB1i) and 5′-UGGUCCUCCUCAAGGCAGU (siNB2i). To test the knockdown specificity and efficiency of the designed siRNAs, HeLa cells stably expressing either HIV-1 Tat or Nullbasic were transfected with siNB1i, siNB2i, and a universal negative-control siRNA (siCTRL) by using Lipofectamine RNAiMAX (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s instructions. Cells were incubated at 37°C for 72 h and then lysed in cell lysis buffer for Western blot analysis. A shRNA, shNB1i, that was based on the siNB1i sequence was constructed. An XbaI site was inserted into the sequence for screening, and the restriction sites HpaI and XhoI were added at the 5′ and 3′ ends, respectively. The shRNA fragments were ligated into pSicoR-Ef1a-mCh between the HpaI and XhoI sites downstream of a U6 promoter. The pSicoR.NBshRNA.mCh vector was confirmed by sequencing. VLPs containing shNB1i were made by cotransfecting HEK293T cells with 10 µg of plasmid pCMVΔR8.91, 3 µg of pCMV-VSV-G, and 3 µg of pSicoR.NBshRNA.mCh. Two rounds of transduction were performed, and flow cytometry showed that >90% of the cells were successfully transduced. The cells were grown for 72 h, after which cells were collected and a lysate was prepared as described previously. The culture supernatant was collected and analyzed for HIV-1 CA by ELISA.

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