Cdna synthesis kit

Total RNAs from choroidal-RPE tissue and cultured ARPE-19 cells were isolated using Trizol reagent. To prevent genomic contamination, RNA was further purified with DNase I (300 ng RNA was treated with 1 μL DNase I at room temperature for 15 minutes and 65°C for 10 minutes after addition of 2.5 mM EDTA). RNA (60 ng) was used to synthesize the cDNA with a cDNA synthesis kit (Exiqon) with deoxy-thymine nucleotide oligomer (oligo-dT) as the primer. The cDNA product (4 ng) was used as a template for PCR reaction with locked nucleic acid (LNA™) primer sets (Exiqon) targeting has-miR-29s and 5 s was used as an internal reference gene. The miRCURY LNA™ SYBR Green was used as reaction dye, and all PCR reactions occurred in a 96-well ABI plate format in ViiA™ 7 real-time PCR system (Applied Biosystems). The relative quantification method (a delta-delta C(T)) was adopted and 40 cycles of PCR reactions (95°C for 10 seconds and 60°C for 1 minute) were started with 50°C for 2 minutes, followed by 95°C for 10 minutes. Dissociative curves were used to confirm the reaction specificity.

The total RNA was isolated using TRIzol reagent as per the manufacturer’s instructions (Thermo Fisher Scientific; Cat. No. 15596026). The cDNA was synthesized from the total RNA using, Universal cDNA synthesis kit II (Exiqon, Vedbaek, Denmark) for miRNA expression analysis or TaKaRa cDNA synthesis kit for mRNA expression analysis. Primers for miRNA-203 (Exiqon), 5S rRNA (Exiqon), BANF1, HBx, HBsAg, HBcAg, HBV-pol and GAPDH (Integrated DNA Technologies, Skokie, IL, USA) (Table 1) were used for real-time PCR using SYBR green in a ViiA 7 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific). Expression analysis of all the genes was done using 2^-ΔΔCt method as described previously (32 (link)).

For evaluating the feasibility of miR-21 inhibition by LNA-anti-miR, reverse transcriptase (RT)-PCR of microRNAs was carried out. After a day of transfection, FavorPrep™ Blood/Cultured Cell Total RNA Kit (Favorgen Biotech Co., Australia) was used based on column chromatography for the extraction of total RNA from the B16F10 cell line. A Nanodrop Epoch (BioTek, USA) was also used to measure the concentration of total RNA. Also, to synthesize cDNA from miRNA, cDNA synthesis kit (Exiqon, Denmark) was employed. To enhance cDNAs from miRNAs and detect miR-21 inhibition, BIOFACT™ 2X PCR master mix (BioFACT™, High ROX, Korea) was used in real-time PCR, along with miR-21-5P and miR-16 specific primers for normalization of real-time PCR; Exiqon (Denmark) provided all primers. Moreover, for 40-cycle real-time PCR, we used StepOnePlus real-time PCR system (Applied Biosystems, USA) was used. Finally, the relative miR21 expression was measured based on 2^−ΔΔCT method.

HeLa R5 cells were transduced at 0.2 MOI with lentivectors carrying the different SMIGs. Transduced population (expressing mCherry) was sorted by FACS resulting in a homogeneous cell population carrying a single copy of the vector per cell. Total RNA was extracted using TRIzol Reagent (Ambion) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop. One hundred nanograms of RNA was used for the reverse transcription (miRCURY locked nucleic acid [LNA] miRNA PCR, polyadenylation and cDNA synthesis kit [exiqon]). Reverse transcription was followed by real-time PCR amplification (ExiLENT SYBR Green master mix kit [exiqon]) with LNA-enhanced primers. Relative expression levels of the mature miRGE were calculated by normalization to the geometric mean of the two housekeeping miRNA (U6 and RNU5G). Fold changes were calculated from the quotient of means of these normalized quantities and reported as ± SEM. Sequences of the LNA-enhanced primers were not provided by the manufacturer.