Mirna qpcr detection kit sybr green

cDNAs of miRNAs and targets were generated from 2 μg of total RNAs of 24 M. hupehensis samples (leaf tissue at 3, 4, 5, 6, 7 and 8 months, and the top leaves of 1-, 2-, 3-, 4-, 5- and 6-year-old trees, as well as roots, stems, flowers and fruit in June) using miRcute miRNA cDNA (Tiangen, Beijing) and PrimeScript™ RT reagent Kit with gDNA Eraser (Takara) (Figure 1). qRT-PCR was performed using a miRNA qPCR Detection Kit (SYBR Green) with 10 μl of 2X miRcute miRNA premix with ROX and SYBR green (Tiangen), and 0.4 μM of forward and reverse primers in a 20-μl system for the expression of miRNAs. PCR was also performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) with 10 μl of 2X SYBR® Premix Ex Taq II, and 0.8 μl of forward and reverse primers in a 20-μl system to determine the expression of the targets (Takara). The reactions were incubated in a Bio-Rad (iCycler iQ5) for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 35 s at 60°C, followed by 81 cycles for the melt curve. Each reaction was performed in three replicates. All primers used in the qRT-PCR experiments are listed in Additional file 2.

For the gene expression assay, total RNA extraction and cDNA synthesis were performed using an RNA extraction kit (TaKaRa, Kusatsu, Japan) and a PrimeScript RT reagent kit (TaKaRa), respectively. Then, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was carried out using the SYBR Premix Ex Taq kit (TaKaRa) with an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, CA, United States). The tomato EF-1α gene served as the reference gene^{71 (link)}. Each qRT-PCR assay was repeated with three biological samples. The relative expression levels of genes were calculated using the 2^–ΔΔCt method.
For the mature miRNA159 expression analysis, the cDNA was generated through reverse transcription from the total RNA by using the miRcute miRNA First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). The qRT-PCR analysis was performed using an miRNA qPCR Detection Kit (SYBR Green) (TIANGEN). The tomato U6 small nuclear RNA gene was used as an internal control. The gene-specific primers used in these procedures are listed in Supplementary Table S4.

In order to validate the sequencing results, qRT-PCR analysis was carried out using selected miRNAs. We extracted total RNA from mouse liver tissue, and reverse-transcripted total RNA into cDNA with miRNA First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). Then the miRNA expression measurement was done by qRT-PCR with miRNA qPCR Detection Kit (SYBR Green) (TIANGEN, Beijing, China) and Roche LightCycler^® 96 fluorescence quantification PCR instrument (Roche, Basel, Switzerland). GAPDH was amplified as internal reference and the relative expression of miRNA to GAPDH was calculated. Primers used in this study were summarized and shown in Table 1.