Sybr green pcr kit

Specific quantitative real-time PCR (qRT-PCR) was set up to estimate the concentrations of microRNA-1 and microRNA-221-3p in the patient and control samples and also microRNA-423-5p as an endogenous control. The reaction components were mixed well and the real-time PCR was performed in a total volume of 10 μl containing 60 ng of cDNA, and 10 pmol of forward and reverse primers using appropriate SYBR Green PCR Kit (Exiqon, Denmark) in a 48-well real-time PCR plate (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions were as follows: 95°C for 10 minutes, 40 cycles at 95°C for 10 seconds, 60°C for 60 seconds, final extension at 72°C for 5 minutes and the cycle threshold (CT) was evaluated, followed by a melting curve analysis. Also, from these tests, CT < 40 was considered suitable to avoid inclusion of non-specific amplification and the mean threshold cycle (ΔCT) of the target gene for the two replicates by fold change method (2^-ΔΔCT) was considered for analysis.^{12 (link)}

Cell/tissue isolated RNA was obtained using the RNeasy kit (Qiagen) and reverse-transcribed with Superscript III (Invitrogen). Quantitative real-time PCR was performed for 45 cycles with the SYBR green PCR Kit [Quiagen] (Beverly, MA, USA) [23 (link)]. Reaction conditions were as follows: 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. Amplification of the target was normalized to the amplification of TATA box binding protein (TBP) and to the levels of an appropriate control using the delta Ct method [55 (link)]. The specific primer sequences (RealTimePrimers.com, accessed 2018-2022) used were as follows: STAT1: Forward, 5′-TCT TGG GAC GTA GCT GAG TG-3′ and Reverse, 5′-GCC GGT AAG AGC TGA GAT TC-3′; STAT3: Forward, 5′-GAC CGT CTG GAA AAC TGG AT-3′ and Reverse, 5′-ACA GAT CCA CGA TCC TCT CC-3′; iNOS: Forward, 5′-TCA GGC TTG GGT CTT GTT AG-3′ and Reverse, 5′-GGAAACCATTTTGATGCTTG-3′; eNOS: Forward, 5′-AGC ACT TGG AAA ATG AGC AG-3′ and Reverse, 5′-CAC TGC ATT GGC TAC TTC CT-3′; IFN-γ: Forward, 5′-CCA AGT TCG AGG TGA ACA AC-3′ and Reverse, 5′-ACT CCT TTT CCG CTT CCT TA-3′; IL-12α: Forward, 5′-CTG CCT CCA CAA AAG ACT TC-3′ and Reverse, 5′-GAT TCA GAG ACC GCA TTA GC-3′; and TBP: Forward, 5′-CGA TAA CCC AGA AAG TCG AA-3′ and Reverse, 5′-AGA TGG GAA TTC CAG GAG TC-3′.

Up to to 2.5 µg of RNA extracted from GL2, IKKαKD and IKKβKD chondrocytes in basal or IL-1β stimulated (8 hours) conditions were reverse transcribed with the Superscript VILO cDNA Synthesis kit (Life Technologies) according to the manufacturer’s instructions. CCL2/MCP-1 messenger RNA (mRNA) was quantified by real-time reverse transcriptase (RT) polymerase chain reaction (PCR) using SYBR Green PCR kit (Quiagen) in a Light Cycler 2.0 instrument (Roche Diagnostics), with values normalized to the expression of GAPDH mRNA^{27 (link)}. Annealing temperature was 56 °C for both GAPDH primers (forward: CGGAGTCAACGGATTTGG; reverse: CCTGGAAGATGGTGATGG) and CCL2/MCP-1 primers (forward: GAAGCTCGCACTCTCGCCT; reverse: GAGTGTTCAAGTCTTCGGA).

Total RNA was isolated from the heart using the RNeasy Lipit Tissue Mini Kit (Qiagen, Inc., Germantown, MD) and further purified using RNeasy MinElute Cleanup Kit (Qiagen, Inc., Germantown, MD) followed by quality assessment on an Agilent 2100 bioanalyzer, as described previously (18 (link), 19 (link)). Relative quantification of mRNA levels by real-time qPCR for ACE, angiotensin converting enzyme 2 (ACE2), mast cell proteases (MCP-1, and MCP-5) was performed using a SYBR Green PCR kit (Qiagen, Inc., Germantown, MD) (19 (link)). Amplification and detection were performed with the QuantStudio 3 real-time PCR Systems (Applied Biosystems, Foster City, CA). Sequence-specific oligonucleotide primers were designed according to published GenBank sequences and confirmed with OligoAnalyzer 3.0. The relative target mRNA levels in each sample were normalized to GAPDH. Expression levels are reported relative to the mean value of the control group.
Cardiac ACE and ACE2 activities were determined, as described elsewhere (20 (link)) in plasma membranes obtained from the hearts of TGR(hAGT)L1623 rats using ¹²⁵I-Ang I and ¹²⁵I-Ang II, respectively. The method described by Ahmad et al. (20 (link)) was employed for determination of chymase enzymatic activities with radiolabeled hAng-(1-12) as the substrate.

Quantitative real-time PCR (qPCR) was performed to validate CNVs. VPS29 gene was selected as the reference gene (endogenous control) for normalization purposes due to the absence of any reported CNV in this gene. Primer were designed, and qPCR using SYBR green PCR Kit (Qiagen, Germany) was performed on a 7500 Real-time PCR System (Applied Biosystems, MA). Quantification of target sequence was normalized, and relative copy number (RCN) determined based on comparative ΔΔCt method with a normal control DNA as the calibrator. The ΔΔCt was calculated as follows: ΔΔCt = (ΔCt unknown sample-ΔCt control sample). Normalized copy number = 2^ˉΔΔCt. A 0.5-fold RCN was selected as a loss (heterozygous deletion), and above 1.5-fold RCN was chosen as a gain [from 3 copies] [25 (link)]. The details of all primer sequences are available in the supporting information (S5 Table in S1 File).