Shrna nc

Since there is no published shRNA sequence that can effectively silence rat NET, we designed and screened the shRNA that can effectively silence the gene. First, the full length of mRNA sequence of SLC6A2 gene of rat was found in NCBI Genebank, and then according to the basic principle of designing shRNA as a template^{35 (link)}, we obtained 4 target sequences of ShRNA-629, 330, 1222 and 1146, which were designed using the Ambion application (Ambion, Austin, TX, USA). Meanwhile, lentivirus expressing shRNA against NET, which were constructed of transfer vector [LV3 (H1/GFP/PURO)] and three helper vectors (pGag/Pol, pRev and pVSV-G), together with nonsense shRNA as negative control (shRNA-NC) were purchased from GenePharma (GenePharma, Shanghai, China)
The shRNA sequences were depicted as below:
ShRNA-629: 5′-GGCGACCATACCAAATACTCC-3′;
ShRNA-330: 5′-CATATACACTGTTCCTCATCA-3′;
ShRNA-1222: 5′-CATTTCTACTCTGTCGGGATC-3′;
ShRNA-1146: 5′-CCCATGAACATAAAGTCAAG-3′;
shRNA-NC: 5′-TTCTCCGAACGTGTCACGTTTC-3′.

All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, with approval of the Ethics Committee of Guangzhou Forevergen Medical Laboratory Animal Center (Guangdong, China). Six Sprague-Dawley rats aged 6–8 weeks (200–250 g) were divided into two groups randomly using a random number table: The sham group and the AKI group. The IRI-induced AKI animal model was constructed by following the protocol of our previous study [18 (link)]. In brief, ischemia was induced by blocking the left renal vertebral arch and arteries for 45 min, and the removal of the right kidney. After 48 h of reperfusion, blood and left kidney tissue were collected for further study.
To investigate whether circ-Snrk had an effect on the IRI-induced AKI animal model, the lentivirus target for circ-Snrk (shRNA), as well as the negative control lentivirus (shRNA-NC), were purchased from Genepharma (Shanghai, China), Then another 15 Sprague-Dawley rats were used for this experiment and randomly divided into three groups (five rats per group): sham group, AKI + shRNA NC group, and AKI + shRNA group. For AKI + shRNA NC group and AKI + shRNA group, lentivirus was injected into tail vein with 1 × 10⁹TU following with previous study [19 ].

A lentiviral vector carrying MEG3 (MEG3 group) and its control lentiviral vector (vector group), as well as a lentiviral vector carrying MEG3-specific short-hairpin RNA (shRNA; MEG3 shRNA group) and its control lentiviral vector (NC shRNA group) were purchased from Shanghai GenePharma Co. Ltd. Cell transfection was performed according to the lentiviral protocol. Siha or HeLa cells in logarithmic growth phase were seeded into 12-well plates at a density of 0.5 × 10⁵ cells/well, and 40% confluent cells were transfected with different groups of lentiviral vectors. After screening with puromycin (1 μg/mL), cervical cancer cells stably expressing high or low level of MEG3 were obtained.