PrS cells were treated with 10 and 50 nM 1,25(OH)2D for 8 or 16 hours (hrs) using ethanol (EtOH) as a vehicle control. RNA was extracted (described above) and cDNA generated from 40ng RNA with the Exiqon Universal cDNA Synthesis kit (Exiqon, Denmark). Exiqon© qPCR array panels I+II (768 miRs) were run according to the manufacturer’s protocol. miR-27b-3p was selected as the optimal reference gene using the normfinder GenX software (Figure S1 ). Per Exiqon guidelines, Ct values greater than 37 were omitted. The full array Ct values are available in Table S1 . Analysis. Relative quantities (RQ) were determined by the ΔΔCt method. logRQ was used to calculated student’s t-test for each miR. miRs with p<0.05 were selected for further analysis. Heat map. RQ values were calculated relative to the EtOH control for each cell line. miRs with p<0.10 (t-test) were imported into GenePattern (Broad Institute, Cambridge, MA, USA) [20 ], log2 transformed and a heatmap created sorted by logRQ at 16h-50nM.
Exiqon universal cdna synthesis kit
Manufactured by Qiagen
Sourced in Denmark
The Exiqon Universal cDNA Synthesis kit is a laboratory equipment product designed for the conversion of RNA to complementary DNA (cDNA). The kit provides the necessary reagents and protocols for efficient and reliable cDNA synthesis, which is a critical step in various molecular biology and gene expression analysis applications.
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2 protocols using exiqon universal cdna synthesis kit
1
1,25(OH)2D Modulates MicroRNA Expression
2
Quantitative miRNA Expression Analysis
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Reverse transcription reactions and real-time PCR analysis were performed using Exiqon universal cDNA synthesis kit (Exiqon, Woburn, MA) and SYBR® Green master mix kit (Exiqon), respectively, in accordance with the manufacturer’s instructions. The cDNA was firstly diluted with RNase-free water at the ratio of 1:40 and then determined by real-time PCR in the StepOnePlus™ Real-Time PCR System (Applied Biosystems®). After amplification, the data was analyzed by the StepOne software v2.2.2. The miRNA abundance was measured based on comparing Ct values of samples to dilutions of a synthetic cDNA of the corresponding miRNA sequence to make a standard curve.
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