Ncode vilo microrna cdna synthesis kit

Small RNA was isolated using PureLink MicroRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion). Poly(A) tailing and cDNA synthesis of the DNAse-treated small RNA were performed using Ncode VILO MicroRNA cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The forward primers for qRT-PCR analysis were designed based on entire known mature microRNA sequence, with additional 3 “A”s at the 3′ end to improve amplification specificity (Table 2). The reverse primer used was the Universal Primer in the EXPRESS SYBR GreenER MicroRNA qRT-PCR Kit (Invitrogen). 5S rRNA was selected as the internal reference gene for PCR quantification. To determine absolute copy number, a standard curve was generated using a synthetic LIN-4 RNA oligonucleotide.
qPCR was performed on iQ5 RT-PCR Detection System (BioRad). All reactions were run in triplicate.

For mRNA qRCR, total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol. First-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). 1 μL of cDNA reaction mix was subjected to PCR amplification using PCR SuperMix (Invitrogen). The forward and reverse primers for qPCR analysis were listed in Table 2. GAPDH was selected as the internal reference gene for PCR quantification.
For miRNA qPCR, small RNA was isolated using PureLink microRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion). PolyA tailing and cDNA synthesis of the DNase-treated small RNA were performed using Ncode VILO microRNA cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The forward primers for qPCR analysis were designed based on entire known mature miRNA sequence, with additional 3 “A”s at the 3′ end to improve amplification specificity (Table 2). The reverse primer used was the universal primer in the EXPRESS SYBR GreenER microRNA qRT-PCR Kit (Invitrogen). 5S rRNA was selected as the internal reference gene for PCR quantification. To determine absolute copy number, a standard curve was generated using a synthetic LIN-4 RNA oligonucleotide.
qPCR was performed on iQ5 RT-PCR detection system (BioRad). All reactions were run in triplicate.

For microRNA qPCR, small RNA was isolated using PureLink microRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion, Austin, TX, USA). Then polyA tailing and cDNA synthesis were performed using Ncode VILO microRNA cDNA Synthesis Kit (Invitrogen). The forward primers for qPCR analysis were designed based on the known mature microRNA sequence, with additional 3 “A”s at the 3′ end to improve amplification specificity (Supplementary Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/3598542). The reverse primer was the Universal Primer in the EXPRESS SYBR Green microRNA qRT-PCR Kit (Invitrogen). To determine absolute copy number, a standard curve was generated using a synthetic LIN-4 RNA oligonucleotide. For mRNA qRCR, total RNA was extracted using TRIzol (Invitrogen) and first-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). 1 μL of cDNA reaction mix was subjected to PCR amplification using PCR SuperMix (Invitrogen). The forward and reverse primers for qPCR analysis were listed in Supplementary Table 1. GAPDH was used as the internal reference gene for relative quantification. qPCR was performed on iQ5 RT-PCR Detection System (BioRad, Berkeley, CA, USA). All reactions were run in triplicate.