Power sybr green rt pcr reagents kit

The number of clean reads per gene per sample was counted using HTSeq version 0.6.0 [15 (link)]. Reads per kilobase per million mapped reads (RPKM) were calculated to estimate the expression level of genes in each sample using the formula as below.
$RPKM=\frac{{10}^6\times R}{\raisebox{1ex}{$N\times L$}\!\left/ \!\raisebox{-1ex}{${10}^3$}\right.}$
Where R is the number of reads in a sample assigned to a gene, N is the total number of mapped reads in the sample, and L is the length of the gene [16 (link)].
Pairwise analyses of differential gene expression were carried out with biological replicates using DEGseq version 1.16. The gene expression levels were determined using a negative binomial distribution model, and genes with q ≤ 0.05 and |log2ratio| ≥ 1 were identified as differentially expressed genes (DEGs) [17 (link)]. The expression levels of all divergent genes were log2-transformed, the Euclidean distance was calculated, and clustering was performed using Hierarchical Cluster methods in R software (version 3.2.5). Quantitative reverse transcription PCR (qRT-PCR) was performed using a Power SYBRGreen RT-PCR Reagents Kit (Applied Biosystems, Carlsbad, CA, USA) in triplicate for each sample and reference gene. Actin was used as a reference gene. Quantification of all chosen lncRNA and mRNA expressions was conducted using the comparative CT ratio between EXOP, StAR with Actin(the primers data showed in S1 File).

To analyze the expression of target mRNAs, total RNA was isolated using the miRNAeasy Mini Kit (QIAGEN, Germantown, MD, USA) and reverse-transcribed to cDNAs using cDNA synthesis kit (OriGene, Rockville, MD, USA). The generated cDNA was amplified using power SYBR green RT-PCR reagents kit (Applied Biosystems, Foster City, CA, USA). The reaction was run at 95 °C for 10 min, which was followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, on a qPCR Instrument (Quantabio, Beverly, MA, USA). The relative transcript expression levels were measured by quantitative real-time PCR using the SYBR Green-based method. The average fold changes were calculated based on 18S in the threshold cycle (Cq). All the primer sequences are in Table 1.