Superscript 3 platinum sybr green one step qrt pcr

Young developing leaves were collected from the same tiller and incubated at the room temperature (25°C) or 42°C for 3 h for the control and the heat‐shock treatments, respectively. The total RNA was isolated from 100 mg samples using the QIAGEN RNeasy plant mini kit (Qiagen, Valencia, CA), and treated with RNase‐Free RQ1 DNase (Promega, San Luis Obispo, CA), and quantified using NanoDrop 2000 (Thermo Fisher Scientific, NY). The expression analysis on Cas9 and sgRNAs was performed on 25 ng of RNA using Superscript III Platinum SYBR green one step qRT‐PCR (Life Technologies, Grand Island, NY) in the CFX96 Real‐Time PCR Detection system (Bio‐Rad, Hercules, CA). The values were normalized against the rice ubiquitin gene, and the relative expression to the non‐transgenic control was calculated using the 2^ΔΔCt (Livak & Schmittgen, 2001) method. Standard errors of two to six biological replicates were calculated. Each biological replicate was repeated two times for the analysis. Student t test (unpaired) was used to determine the p‐value. Primers used in qRT‐PCR are given in Table S6.

Liver tissue from animals in each group was collected, immediately frozen in liquid nitrogen, and stored at −80 °C until RNA extraction was performed. For the extraction of total RNA, TRIZOL™ reagent (Life Technologies Carlsbad, Carlsbad, CA, USA) was used according to the protocol proposed by the manufacturer.
The RNA concentration was determined by the NanoDrop ND-1000 spectrophotometer. The degree of RNA purity was evaluated by a 260/280 nm ratio, using only those whose ratio was ≥1.8. The integrity profile of extracted RNA was evaluated by electrophoresis to verify the presence of bands corresponding to 18S and 28S ribosomal RNAs. The quantified RNA was stored at −80 °C until use.
The design of oligonucleotides was conducted with the Primer 3 program (http://primer3.ut.ee accessed on 22 September 2022). Analysis of the expression of mRNA levels of BAX, Bcl-2, CASPASE 3, and CASPASE 8 genes was performed on a Rotor-Gene RG-3000 thermocycler (Corbett Research, Sydney, Australia). The commercial kit SuperScript™ III Platinum^® SYBR Green One-Step qRT-PCR (Life Technologies Corporation, Carlsbad, CA, USA) was used. The beta-actin gene was used as a normalizer of qRT-PCR reactions. The 2-Delta Delta CT method was used for relative quantification of gene expression (Livak & Schmittgen, 2001).

The ADSCs were seeded in 75 cm² culture flasks and divided into three groups: stimulated with Col V (50 μg/ml) for 72 h; TGF-β1 (10 ng/ml) for 72 h, a growth factor widely used to induce cellular differentiation; and without stimulus. Total RNA was extracted by the method of Chomczynski and Sacchi (1987) (link) using the TRIzol reagent and RNA samples were treated with the kit RQ1 RNase-Free DNase (Promega^®, Thermo Fisher Scientific, Rockford, IL), according to the manufacturer’s instructions. RT-PCR reactions were performed with the primers drawn to Col2a1 (α1 chain Col II), Acan (aggrecan), and Pou5f1 (POU domain, class 5, transcription factor 1, protein transcription factor involved in the process of self-renewal and maintenance of stem cells) (Table 1).
Gene expression was evaluated using the Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) with the kit SuperScript III Platinum SYBR^®, Green One-Step qRT-PCR (Life Technologies). The cycling conditions for the genes were as follows: 50°C for 10 min (for cDNA synthesis) followed by 35 cycles of 95°C for 15 s, 60°C for Gapdh and Col2a1, 59°C for Acan, and 57°C for the Pou5f1 gene for 30 s and 72°C for 30 s. The analysis was performed by the 2^–ΔΔCT method using Gapdh gene as an internal control.