miRNA were extracted from fresh frozen colon adenocarcinoma and normal colon tissues using miRNeasy mini kit (Qiagen) according to manufacturer’s instructions. RNA concentrations were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using miScript II RT Kit (Qiagen) in accordance with the manufacturer’s instructions.
Expression of miR-205 and miR-338-5p was determined by real time PCR using an Absolute SYBR Green Rox mix (Thermo Fisher Scientific), on a CFX 96 real time PCR detection system (Bio-Rad) and normalized using U6 small nuclear RNA. miR-205 is known to down-regulate LRP1 expression [42 (link), 43 (link)]. miR-338-5p is not implicated in LRP1 expression regulation and was used as control as previously described [43 (link)]. All primers were purchased as 10x miScript Primer Assay (Qiagen). PCR conditions were 15 min at 95° C, followed by 40 cycles each consisting of 15 s at 95° C (denaturation), 30 s at 55° C (annealing) and 30 s at 70° C (extension). The specificity of PCR amplification was checked using a heat dissociation curve from 65° C to 95° C following the final cycle. The cycle threshold (Ct) values were recorded with Bio-Rad CFX Manager™ 3.0 software (Bio-Rad).
Expression of miR-205 and miR-338-5p was determined by real time PCR using an Absolute SYBR Green Rox mix (Thermo Fisher Scientific), on a CFX 96 real time PCR detection system (Bio-Rad) and normalized using U6 small nuclear RNA. miR-205 is known to down-regulate LRP1 expression [42 (link), 43 (link)]. miR-338-5p is not implicated in LRP1 expression regulation and was used as control as previously described [43 (link)]. All primers were purchased as 10x miScript Primer Assay (Qiagen). PCR conditions were 15 min at 95° C, followed by 40 cycles each consisting of 15 s at 95° C (denaturation), 30 s at 55° C (annealing) and 30 s at 70° C (extension). The specificity of PCR amplification was checked using a heat dissociation curve from 65° C to 95° C following the final cycle. The cycle threshold (Ct) values were recorded with Bio-Rad CFX Manager™ 3.0 software (Bio-Rad).
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