Mircury lnatm detection probe

A 5′-DIG and 3′-DIG-labeled, locked nucleic acid-incorporated miRNA probe (miRCURY LNATM Detection probe, Exiqon, Woburn, MA, USA) was applied for the detection of miR-323a-3p in BCa TMAs. The probe sequence of hsa-mir-323a-3p was designed as follows: 5′-AGAGGTCGAC CGTGTAATGTG-3′. The specific manipulations were performed as previously described.^{48 (link)} Both the intensity and proportion of positive cells were considered for the semi-quantification of the strength of positivity.

For IHC analysis of SIRT1, 4 μm paraffin-embedded tissue microarray sections were incubated overnight at 4°C with primary antibodies against human SIRT1 (13161-1-AP, proteintech; 1:50 dilution). Sections were then washed and incubated for 30 min with biotinylated goat anti-rabbit IgG, washed thoroughly, and stained with diaminobenzidine. For ISH analysis of miR-138-5p, paraffin-embedded tissue sections were treated with miRCURY LNAtm Detection probe (Exiqon, Vedbaek, Denmark), following the manufacturer's protocol. Images were captured using an EnVision+ detection system (Dako, Wuhan, China). Two pathologists blinded to the patient's diagnosis and outcome examined each sample. Staining intensity and the proportion of positive cells were recorded.

Tissue microarrays (TMAs) were purchased from Xinchao Biotech, Shanghai, China, which contained 81 cases with paired tumor and non-tumor tissues. A 5′-DIG and 3′-DIG-labeled, locked nucleic acid-incorporated miRNA probe (miRCURY LNATM Detection probe, Exiqon, Woburn, MA, USA) was applied for the detection of miR-493-3p in PCa TMAs, The probe sequence of hsa-miR-493-3p was designed as follows: 5’-CCTGGCACACAGTAGACCTTCA -3’. The specific procedures were performed as previously described [34 (link)]. Both the intensity and proportion of positive cells were considered for the semi-quantification of the strength of positivity.