Faststart universal sybr master

Relative quantification of mRNA expression was conducted by RT-qPCR, using the FastStart Universal SYBR Master (Roche Applied Science) reaction mix. First strand cDNA synthesis was performed with 1 μg of total RNA using 0.5 μg oligo (dT)₁₅ primer (Promega) and SuperScript^® II Reverse Transcriptase (Invitrogen) in a 20 μl final volume using the manufacturer’s recommended standard protocol.
RT-qPCR analysis was performed using a Rotor-Gene™ Q (Qiagen, Crawley, UK) with reactions prepared using a CAS-1200™ Precision Liquid Handling System (Qiagen). All reactions were performed in final volume of 15 μl and ‘no template’ controls were included for each primer pair. Gene specific primers sequences and PCR conditions for qPCR analysis are listed in Table S1. The amplification profile consisted of 10 min at 95 °C, followed by 40 cycles of gene-specific cycling conditions (Table S1), followed by a dissociation curve analysis. The cycle threshold value (Cq) was determined using the Rotor-Gene Q Software version 2.3.1 (build 49).
Agarose gel electrophoresis of amplicons confirmed a single product and sequence analysis was used to confirmed specificity of primer pairs. The linearity and efficiency of RT-qPCR amplification was determined for each primer pair (Table S1) using a standard curve generated by a dilution series of a pool of sample cDNAs.

The following reagents were purchased from Roche: FastStart Universal SYBR Green Master ROX (2×) kit (Roche Cat# 4913914001), Tripure isolation reagent (Cat# 11667165001), FastStart Universal SYBR Master (Cat# 04913850001), and In Situ Cell Death Detection Kit (Cat# 11684817910). The ReverTra Ace quantitative real-time polymerase chain reaction (qPCR RT) kit was obtained from TOYOBO (Cat# FSQ-101). The Animal Total RNA Rapid Extraction Kit was from JieRui (Cat# GK3016). The RevertAid First Strand cDNA Synthesis Kit (K1622) was from Thermo Scientific (Cat# 1622). The following measurement kits were purchased from Nanjing Jiancheng Bioengineering Institute: superoxide dismutase (SOD) (Cat# A001-1), catalase (CAT) (Cat# A007-1), malondialdehyde (MDA) (Cat# A003-1), nitric oxide synthase (NOS) (Cat# A014-2), and NO (Cat# A012). Rabbit-anti-mouse NF-κB antibody was from Abcam (Cat# Ab32536). The immunohistochemistry (IHC) and enhanced DAB chromogenic kits were obtained from Mai Xin.

ChIP assay was performed as previously described [9 (link)]. Briefly, LNCaP cells were cross-linked with paraformaldehyde and digested with micrococcal nuclease to achieve a DNA smear of 200-1000 bp. ChIP assay was performed using SimpleChIP^TM Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's protocol on the HER2 or PSA gene. The quantitative RT-PCR assay with DNA extraction, the primer pairs below and FastStart Universal SYBR Master (Roche, Missassauga, ON) was performed using ABI 7900HT System (Applied Biosystems). The results are representative of at least three independent experiments. The sequences of primer pairs are as follows: 5′-AGGGGCTCCAAATAGAATGT-3′ (Fw) and 5′-AATTTGGGAGGAGACAGTCA-3′ (Rv) for HER2 promoter targeting between –978 bp and –514 bp from transcription start site (TSS) [43 (link)]; 5′-TCTGCCTTTGTCCCCTAGAT-3′ (Fw) and 5′-AACCTTCATTCCCCAGGACT-3′ (Rv) for PSA ARE+ targeting between –250 bp and –39 bp from TSS; and 5′-CTGTGCTTGGAGTTTACCTGA-3′ (Fw) and 5′-GCAGAGGTTGCAGTGAGCC-3′ (Rv) for PSA ARE– targeting between –1997 bp and –1846 bp from TSS.

Total RNA was extracted with TRIzol (Thermo Fisher, MA, USA) following procedures recommended by the manufacturer. One microgram of RNA was reverse transcribed using the Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany), and 1 μl of the cDNA was used in a qPCR with the FastStart Universal SYBR Master (Roche, Basel, Switzerland). Concentrations of SaCas9 and HBV RNA were determined relative to mRNA of GAPDH, while the sgRNAs were measured relative to hU6snRNA using primers described in Supp. Table 4.