Mircury lna universal rt microrna pcr starter kit

RNA concentration was assayed using the Qubit^® Fluorometer (Molecular Probes by Life Technologies) using the Qubit^® RNA HS Assay Kits (Q32852 Molecular Probes), according to the manufacturer’s recommendations. During protocol development, quality and concentration were also evaluated by electrophoretic assays on an Agilent Bioanalyzer 2100, using standard procedures.
In addition, RT-qPCR was used to quantify the expression of miRNA-125b-5p, which has been shown to be highly expressed in sperm [41 (link)]. Total RNAs (5 ng) were reverse transcribed in 10-μl reactions using the miRCURY LNA™ Universal RT microRNA PCR Starter kit (Exiqon). RNA spike-in control (Usp6) was also added during the RT-step. Triplicates were assayed in 10-μl qPCR reactions following the miRCURY LNA kit protocol, using a StepOnePlus Real time PCR System (Applied biosystems). Amplification curves were analyzed using the StepOne software v2.3 both for determination of Ct values and for melting curve analysis.
Library preparation and sequencing was done by the Exiqon service provider. Briefly, miRNA libraries were constructed using the NEBNext^® Multiplex Small RNA Library Prep Set for Illumina^® kit, including a size selection and quality control by Bioanalyzer. Deep sequencing was performed on Illumina HISeq2000, targeting 40 M single-reads (50 bp) per sample.

DLD-1 cells (1 × 10⁶ cells/well) were seeded into 100 mm cell culture plates and stabilized for 24–36 h. IL-17C (100 ng/mL) was treated to the cultured cells for 3 h, and total mRNA was isolated using miRCURY miRNA isolation kits (Exiqon, Woburn, MA, USA). To quantify miRNA transcript levels, qPCR was performed using the miRCURY LNA™ Universal RT microRNA PCR Starter Kit (Exiqon) and miScript Primer Assay (Qiagen), following the manufacturer’s instructions. The primer sequences used to detect miR-23a-3p and the endogenous control miR-103a-3p were as follows: hsa-miR-23a-3p (5′- AUC ACA UUG CCA GGG AUU UCC -3′); hsa-miR-103a-3p (5′- AGC AUU GUA CAG GGC UAU GA -3′).

To conduct the quantitative reverse transcription PCR (RT-qPCR) for the detection of miR-663a expression level, complementary DNA (cDNA) synthesis from miRNA was performed using miRCURY LNA™ Universal RT microRNA PCR Starter Kit (EXIQON, Vedbaek, Denmark), according to manufacturer’s instructions. Then, real-time PCR amplification was performed in a 10 μL solution containing PCR master mix, PCR primer mix (EXIQON, product no: 204284), and cDNA template. Similarly, cDNA synthesis from total RNA was carried out using PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKaRa Bio, Shiga, Japan) and RT-PCR was performed in a 12.5 μL solution containing 2 × SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKaRa Bio), 1 μL cDNA, and 0.4 μM of each primer. The sequences of the primers are available in Additional file 1: Table S1). Each RT-qPCR assay was performed in triplicate using the Thermal Cycler Dice Real Time System Single (TaKaRa Bio). Relative expression levels were calculated by the ΔΔCt method. The expression levels of target genes such as miR-663a and other genes were normalized to U6 and GAPDH, respectively.