Mir p081

The pLV-miRNA vector, comprising hsa-mir-106b lentivirus and co-expressing GFP protein in infected E. coli BL21 was purchased from Biosettia Inc. (mir-p081, Biosettia, San Diego, CA, USA). Transfection of SSCs was done by 2.5 μg of the pLV-miRNA vector. For this reason, mouse SSCs (1.0 ×10 6 ) were seeded in a 6-well plate before transfection so that the cell density was around 70%-90%. Gently, 500 μl of culture medium and 7.5 μl of Lipofectamine3000 (Invitrogen, USA) solution were mixed and incubated for 10-15 minutes at room temperature. Meanwhile, 2.5 μg of the pLV-miRNA vector was added to 500 μl of the medium. Then, 250 ml of the produced solution was added to each well and the cells were incubated at 37 ° C for 2-4 days. In order to con rm the transfection e ciency and observe the expression of the GFP gene under a uorescence microscope, the quantitative real-time reverse transcriptase PCR (qRT-PCR) technique was used. For this purpose, the sequences of forward and reverse primers, mir-106b, and U6 snRNA (as a reference gene) were downloaded from the www.ncbi.nlm.nih.gov/Gene website and designed using GeneRunner software (Table 1). The cDNA was synthesized according to the manufacturer's protocols (Fermentas, USA) and the products were analyzed by electrophoresis in 1% agarose gel with a DNA ladder (1kb).