A single nucleotide polymorphism in a miR-1302 binding site in CGA increases the risk of idiopathic male infertility  Hao Zhang, Ph.D., Yunqiang Liu, Ph.D., Dan Su, Ph.D., Yuan Yang, M.D., Gang Bai, Ph.D., Dachang Tao, Ph.D., Yongxin Ma, M.D., Sizhong Zhang, M.D.  Fertility and Sterility  Volume 96, Issue 1, Pages 34-39.e7 (July 2011) DOI: 10.1016/j.fertnstert.2011.04.053 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A–C) Predicted effect of allelic variation at rs6631 on the recognition of miR-34c-5p (A) miR-610 (B) and miR-1302 (C) to CGA. The variation site is labeled with red color. (D) Reverse transcriptase-polymerase chain reaction detects the expression of miR-34c-5p (A) miR-610 (B) and miR-1302 (C) in HEK293T cells (23). M = PUC19/MspI marker; NC = negative control. Fertility and Sterility 2011 96, 34-39.e7DOI: (10.1016/j.fertnstert.2011.04.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The effect of allelic variation of rs6631 on luciferase activity. (A) Structure of luciferase reporter constructs containing CGA 3′UTR. CGA sequence from +275 to +293 which is deleted in CMV-Luc CGA-Del is represented by the red line; (B) representative graph of luciferase activity of CMV-LUC, CMV-LUC CGA-Del, CMV-LUC CGA-A, and CMV-LUC CGA-T in HEK293T cells. The cells were cotransfected with CMV-LUC, CMV-LUC CGA-Del, CMV-LUC CGA-A, or CMV-LUC CGA-T and PRL3-CMV; (C) inhibition of miR-1302 results in similar luciferase activity. HEK293T cells were cotransfected with CMV-LUC CGA-T, CGA-A, or CGA-Del and PRL3-CMV Renilla with either miR-1302 inhibitor or a negative control miRNA. Values are means of firefly/Renilla ratio from four independent experiments. Error bars indicate 1 SD of these experiments. P values were calculated by independent samples t-test. Triplicated repeats were conducted showing similar results. Fertility and Sterility 2011 96, 34-39.e7DOI: (10.1016/j.fertnstert.2011.04.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions