Sybr green 1 pcr master mix

RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). The cDNA was used as a template in quantitative real-time PCR reactions performed using SYBR green I PCR Master Mix and an ICycler system (Bio-Rad). The following primers were designed from the full-length AR and Nox4 mRNA sequences and synthesized by SBS Biotechnology Corporation (Beijing, China): AR sense, 5′- CCTATGGCCAAGGACACACT-3′ and antisense, 5′-CTGGTCTCAGGCAAGGAAAG-3′; NOX4 sense, 5′-TTGCCTGGAAGAACCCAAGT -3′ and antisense, 5′- TCCGCACAATAAAGGCACAA-3′. As an internal control, mouse GAPDH was amplified using the following primers: sense, 5′-GGCATGGACTGTGGTCATGAG-3′ and antisense, 5′-TGCACCACCAACTGCTTAGC-3′. Relative expression (fold change vs. control) was quantified using the 2^-ΔΔCt method.

Total RNA was extracted using the TRIzol method (Invitrogen Life Technologies) according to the manufacturer's instructions and reverse-transcription was subsequently performed with a reverse transcription kit (Invitrogen).
Each fragment was amplified in a 20 μl reaction containing 1X SYBR Green I PCR Master mix (Bio-Rad Laboratories), 20 pmol of each primer pair, 20 ng of cDNA (total RNA equivalent), and nuclease-free water. The thermal profile consisted of 1 cycle at 95°C for 3 min followed by 45 cycles at 95°C for 10 s, 60°C for 10 s, 72°C for 20 s. The PCR reaction was followed by a melting profile analysis. A glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragment was amplified as the internal control for the amount of cDNA in the qPCR. Each experiment was performed in triplicate.
The primers sequences were as follows for human samples and for murine samples:
Human samples:
FTH for 5′-CATCAACCGCCAGATCAAC-3′
FTH rev 5′-GATGGCTTTCACCTGCTCAT-3′
GAPDH for 5′-TGATGACATCAAGAAGGTGGTTGAAG-3′
GAPDH rev 5′-TCCTTGGAGGCCATGTGGGCCAT-3′
HLA-I for 5′-CCTTGTGTGGGACTGAGAGG-3′
HLA-I rev 5′-CAGAGATGGAGACACCTC-3′
Mouse samples:
GAPDH for 5′-AACACCACCATGGAGAAGGC-3′
GAPDH rev 5′-ACAGCCTTGGCAGCACCAGT-3′
FTH for 5′-CATCAACCGCCAGATCAAC-3′
FTH rev 5′-GATGGCTTTCACCTGCTCAT-3′
FTL for 5′-CTTGCCCGGGACTTAGAGCA-3′
FTL rev 5′-ATGGCTGATCCGGAGTAGGA-3′

Total RNA was prepared using the RNeasy Mini Kit (Qiagen Inc, Valencia, CA. USA). RNA samples underwent DNase I treatment (Promega, Madison, WI, USA) prior to first-strand cDNA synthesis with random hexamer primers using the Superscript II First-Strand cDNA Synthesis System (Invitrogen, Grand Island, NY, USA). Real-time PCR was carried out using the ABI 7300 model sequence detection system (Applied Biosystem, Foster City, CA, USA) with SYBR Green I PCR Mastermix (BioRad, Hercules, CA, USA). Primers for ER and EP300 were published [51 (link)–53 (link)]. Primers for BP1 were designed using Primer-BLAST and are as follows: 5′-CCTCCCCCAATTTGTCCTACTC-3′ (forward) and 5′-GGTTGCTGGCAGGACAGGTA-3′ (reverse). The amplification program included an initial denaturation at 95°C for 10 min., followed by 40 cycles of a two-stage PCR consisting of 95°C for 15s and 60°C for 1 min. Specificity for PCR amplifications was verified by observing a single peak dissociation curve for each gene. One microliter of the reverse transcribed cDNA was used for each real-time PCR reaction and all reactions were performed in triplicate. The expression values of genes from different samples were calculated by normalizing with 18S RNA and relative quantitation values were plotted.

In order to detect the effect of tFNAs–miR‐26a on the expression of osteogenic‐specific genes, we used real‐time polymerase chain reaction (RT‐PCR) technology to detect the expression of osteopontin (OPN), alkaline phosphatase (ALP), and runt‐related transcription factor 2 (RUNX2). As shown in Table 2, we also detected the expression of genes related to the Wnt signaling pathway, such as β‐catenin, glycogen synthase kinase (GSK) and lymphatic enhancement factor‐1 (Lef‐1). After treatment with tFNAs–miR‐26a for 1 day, TRIzol reagent (Thermo Fisher Scientific, MA) was employed to extract gene samples. Subsequently, we used a cDNA synthesis kit (Mbi, Glen Burnie, MD) to purify and obtain the cDNA, and then we employed SYBR Green I PCR master mix and the Bio‐Rad real‐time PCR system (Bio‐Rad, Hercules, CA) to perform qPCR.

Biofilm DNA was extracted using the Power Biofilm DNA Isolation kit (Mo Bio Laboratories, USA) according to the manufacturer’s instructions. The abundance of bacterial 16S rRNA, archaea bacteria 16S rRNA, periplasmic nitrate reductase (napA) and membrane-bound nitrate reductase (narG), nitrite reductase (nirS and nirK) and nitric oxide reductase (qnorB) genes were identified in the DNA samples from three biological replicates of each experiment set. Quantitative polymerase chain reaction (qPCR) using a MyiQ2 real-time PCR Detection System (Bio-Rad) with the fluorescent dye SYBR-Green approach was employed using the 16S rRNA gene and functional gene amplification. The amplification of qPCR was performed in 20 μL reaction mixtures containing 10 μL of SYBR Green I PCR master mix (Bio-Rad), 1 μL of template DNA (sample DNA or plasmid DNA for standard curves), 0.5 μL of forward and reverse primers, and 8 μL of sterile water. The qPCR protocol included 40 cycles. The primer sequences and qPCR protocols are provided in Table S1.