Dynamo flash sybr green qpcr kit

Cells were treated as indicated, and total RNA was extracted using guanidine isothiocyanate-phenol-cholorofrom (TRIZOL). RNA concentration was assessed spectrophotometrically using the Nano Drop ND-1000 Spectrophotometer (Thermo Scientific). The ratio of absorbance at 260/230 and at 260/280 was used as indicators for RNA purity. RNA was reverse transcribed to generate cDNA using a Verso cDNA Synthesis Kit (Thermo Scientific). For semi-quantitative analysis, cDNA was amplified using a Taq PCR core kit using gene specific primers. Quantitative real-time PCR was carried out using a CYBR green master mix (DyNAmo Flash SYBR Green qPCR Kit) following manufacturer’s instruction on the CFX96TM real-time system (Biorad, Hercules, CA, USA). All quantifications were performed with 18S RNA as internal control and the relative amount of mRNA was presented in the form of fold change over control. The primers used in the study were designed and validated using Universal Probe Library. The concentration of the primer in the reaction was 1μM and the length of the product ranges from 70–80 bp. The expression data were calculated on the basis of ct value obtained from quantitative real-time PCR. The primer sequences used are shown in Table 1.

Reverse transcription was performed with 500 ng of total RNA using Maxima First Strand cDNA Synthesis Kit for RT–qPCR (Thermo Fisher). The qRT–PCR was performed using DyNAmo Flash SYBR Green qPCR Kit on Bio‐Rad CFX96 Real‐Time System. ACTB and Actb were used as reference genes for human and mouse targets, respectively. Primer sequences are provided in Appendix Table S2. MicroRNA amplification was performed using LNA primers for MIR9 (Exiqon, #204513), and SNORD49A (Exiqon, #203904) was used as a reference gene.