Rna purification kit

Total RNA of the collected glioma tissues and normal tissue samples was extracted by TRIzol kit (Accurate Biology, China, AG21101) and RNA purification kit (Thermo Scientific, K0731), reverse transcribed to cDNA by RT-qPCR kit (Thermo Scientific, K16225). Amplification was performed according to the SYBR green (Bio-Rad, #1725274) method with three replicate wells per sample in a total reaction system of 20 μl. PCR reaction conditions (ABI 7300 Real-Time System) were as follows: 95°C for 15 min, 40 cycles of amplification at 95°C for 15 s, 60°C for 1 min. β-Actin was used as an internal reference and 2^-ΔΔCt formula was used to calculate the relative expression of the targeted gene. Targeted gene primer sequences were as follows:MLLT11: (forward) 5’-GTAGCCAGTACAGTTCCTTTCT-3’, (reverse) 5’-AAGTTGAAGGTGCTGTACTCAA-3’; ARG1: (forward) 5’-GGACCTGCCCTTTGCTGACATC-3’, (reverse) 5’-TCTTCTTGACTTCTGCCACCTTGC-3’; CD206: (forward) 5’-TCCGACCCTTCCTTGACTAATCCTC-3’, (reverse) 5’-AGTATGTCTCCGCTTCATGCCATTG-3’; IL-10: (forward) 5’-GTTGTTAAAGGAGTCCTTGCTG-3’, (reverse) 5’-TTCACAGGGAAGAAATCGATGA-3’; CD163: (forward) 5’-ATCAACCCTGCATCTTTAGACA-3’, (reverse) 5’-CTTGTTGTCACATGTGATCCAG-3’; CD115: (forward) 5’-GTCCTGAAGGTGGCTGTGAAGATG-3’, (reverse) 5’-GCTCCCAGAAGGTTGACGATGTTC-3’; PDGFβ: (forward) 5’-TCTCTGCTGCTACCTGCGTCTG-3’, (reverse) 5’-AAGGAGCGGATCGAGTGGTCAC-3’.

The RNA was extracted from liver tissues; pure RNA was obtained using the RNA Purification Kit (Thermo Scientific, Fermentas, #K0731). Complementary DNA (cDNA) was obtained using Revert Aid H minus Reverse Transcriptase, a genetically adjusted M-MuLV RT that converted the RNA into complementary DNA (cDNA). SYBR Green–real-time PCR assay was used to assess the expression of the mRNAs of the liver and testes genes with internal references to β-actin (Table 1).

Leaves were harvested at 2 days after pathogen inoculation. RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA USA) was used to extract RNA. The extracted RNA was converted to complementary DNA (cDNA) using revert Aid H minus reverse transcriptase. Real-time PCR with SYBR Green was used to measure the expression of mRNAs of the target gene (CaPR4, a gene associated with defense response and cell death), with CaActin as an internal reference as described by Elsharkawy and El-Khateeb [9 (link)].

Total RNA was isolated using an RNA Purification Kit (Thermo Fisher Scientific). Total RNA (200 ng) was treated with RNase-free DNase (Sigma Aldrich) for 15 min. Following the inactivation of DNase with Ethylenediaminetetraacetic acid (EDTA) and heating, RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real time polymerase chain reaction (RT-PCR) (qPCR) was performed with cDNA samples using the Power SYBR Green Master Mix and Mic qPCR Cycler (Bio Molecular Systems). The relative mRNA level was calculated as values of 2^{(Ct(β-actin) − Ct(gene of interest))}. For data presentation, the mRNA level in control cells was set to 1. The sequences of the forward and reverse primers are as follows: β actin, 5’-GGCTGTATTCCCCTCCATCG-3’ and 5’-CCAGTTGGTAACAATGCCATGT-3’; Ccna1, 5’-TGATGCTTGTCAAATGCTCAGC-3’ and 5’-AGGTCCTCCTGTACTGCTCAT-3’; Ccna2, 5’-GCCTTCACCATTCATGTGGAT-3’ and 5’-TTGCTGCGGGTAAAGAGACAG-3’.

Total RNA was extracted from the frozen heart LV muscles of normal control, CHF, and drug treated dogs using a Trizol Plus RNA Purification kit (Thermo Fisher Scientific). Total RNA from HCF was extracted using an RNA Purification kit (Thermo Fisher Scientific). For RNA-Seq, total RNA was quantified by an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) following the manufacturer’s instructions. The RNA Integrity Numbers of all prepared total RNA samples were ≥ 8.