Transstart top green qpcr kit

Sediment DNA and RNA were extracted with PowerSoil DNA Isolation Kit and PowerSoil Total RNA Isolation Kit (MoBio, United States), respectively, and were evaluated with Nanodrop 2000 (Thermo Fisher Scientific, United States). RNA further was prepared at a similar concentration before further analysis. Real-time PCR of pmoA gene was performed on a CFX Connect cycler (Bio-Rad, United States), using the primers A189f/mb661r following the PCR conditions reported by Liu et al. (2015) (link). Reactions were proceeded using TransStart Top Green qPCR Kit (Transgen Biotech Co., Ltd, China). Gene transcripts were quantified in a one-step RT-qPCR using a TransScript Green One-step qRT-PCR Kit (Transgen Biotech Co., Ltd, China). Possible gDNA contamination was removed with DNase before reverse transcription. Melting curve analyses were carried out at the end of PCR run to verify the amplification specificity. Each measurement was carried out with three technical replicates, and result was given in average ± standard deviation. Standard curve was constructed with serially diluted plasmids containing pmoA gene fragment, and the efficiency and r-square were 91.5% and 0.998, respectively.

To assess the reliability of the RNA sequencing-based approach in identifying DGEs, qRT-PCR was employed to detect gene transcript patterns. One microgram of total RNA from the mycelia and primordia was adopted to synthesize cDNA using TransScript All-in-One First-Strand cDNA Sythesis Super Mix for qPCR kit (TransGen Biotech, Beijing) in accordance with the manufacturer's protocol. The primers () used for quantitative real-time PCR (qRT-PCR) analysis were designed with primer premier 5.0 [42 (no link found, link)]. Twenty microliters of qRT-PCR reaction mixtures were prepared according to the manufacturer's instructions using TransStart TOP Green qPCR kit (TransGen Biotech, Beijing). The cycling parameters were as follows: 94°C for 30 s followed by 30 cycles of 94°C for 5 s and 60°C for 30 s. Three independent biological replicates were carried out for each gene. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and α-tubulin severed as an internal control gene, and the 2^−ΔΔct method [43 (link)] was applied to the calculated gene expression.