Pcp cy5

All 1921 human mature miRNAs in the miRBase database (Release 18.0) were used for designing probes for constructing the in-house miRNA microarray and a total of 1849 probes have been successfully designed according to the principle proposed by Wang [44 (link)]. The microarray was fabricated in house and hybridized as described by us previously [45 (link), 46 (link)]. Briefly, each probe was mixed with printing buffer to a final concentration of 40 μmol/L and printed in duplicate on the cleaned glass slides (75 × 25 mm). The total RNA (1.0 -1.5 μg) was labeled with 100 nmol/L of pCp-Cy5 (Jena Bioscience, Germany) and 15 units of T4 RNA ligase (USB) in a total reaction volume of 20 μL at 16°C overnight. Then the mixture of labeled RNA sample and 1x hybridization solution was hybridized onto the microarray for 12 -18 h at 45°C. After hybridization, the slides were washed in 1×SSC/1% SDS for 10 min at 45°C, followed by sequential washing in 2 cycles of 0.5 ×SSC/0.1% SDS, 2 cycles of 0.2×SSC and 1 cycle of purified water for 1 min at room temperature, respectively, and then dried in a special small centrifuge and scanned using the LuxScan-10K (CapitalBio, China).

Nucleic acid substrates were prepared by PCR with Cy3/Cy5 conjugated oligos (IDT) as primers (dsDNA), ordered directly as Cy5-conjugated oligos (IDT) (ssDNA), or in vitro transcribed from PCR templates and labeled with pCp-Cy5 (Jena Biosciences) using T4 RNA ligase 1, ssRNA ligase (High Concentration) (NEB) (RNA). Substrates were mixed with protein and buffer components and incubated at various temperatures, and results were resolved by gel electrophoresis, as specified in Materials and Methods.

For detection and quantification on the microarray, all RNA probes in the library were labeled with fluorescent dye at the 3′ end by mixing 1× T4 Ligase Buffer (Thermo Fisher Scientific), 100 μM pCp-Cy5 (Jena Bioscience), 10 μM RNA structure library, and 0.5 U/μL T4 RNA Ligase (Thermo Fisher Scientific) in a 100 μL reaction mixture. The mixture was incubated at 16 °C for 48 h under light-shielded conditions. The labeled RNA probes in the library were purified with Zymo RNA Clean and Concentrator (Zymo Research). The fluorescently labeled RNA structure library was stored at −28 °C.

Cy3- and Cy5-labelled RNAs were prepared by ligation using T4 RNA ligase (Ambion), pCp-Cy3 (Jena Bioscience) and pCp-Cy5 (Jena Bioscience). Cy3- and Cy5-labelling was performed with 150 pmol RNA using 10 U T4 RNA ligase, 3 nmol pCp-Cy3 or pCp-Cy5 and 10% (v/v) dimethyl sulfoxide in 10 µl at 16 °C for 36–48 h. The Cy3- and Cy5-labelled RNAs were purified using an RNeasy MinElute Cleanup Kit (Qiagen). After the recovery of RNAs, the RNA concentration was measured in a NanoDrop (Thermo Scientific). The labelling efficiencies of Cy3 and Cy5 were calculated from the absorbance of Cy3 and Cy5, respectively.