Sparkeasy improved tissue cell rna kit

Total RNA was prepared from AML cells by using a SPARKeasy Improved Tissue/Cell RNA kit (SparkJade, Jinan, China) according to the manufacturer’s protocol. RNA yield was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Complementary DNA was synthesized using a SPARKscript II RT Plus Kit (SparkJade, Jinan, China). Quantitative polymerase chain reaction (qPCR) assays were performed using a 2× SYBR Green qPCR Mix (SparkJade, Jinan, China) in a 20 μL reaction volume, by Roche QRT-PCR System according to the manufacturer’s protocol. The messenger RNA (mRNA) levels of target genes were normalized to the mRNA level of β-actin. The sequences of the primers used for the APOBEC3G were: forward, 5’-TTGCCCGCCTCTACTACTTCTGG-3’ and the reverse, 5’-CTTGCTCCAACAGTGCTGAAATTCG-3’ (Sangon Biotech, Shanghai, China). The cycling conditions used were: initial denaturation at 94 °C for 3 min, followed by 40 cycles at 94 °C for 10 s, 60 °C for 30 s.

We extracted total RNA from cells in each group with a SPARK easy Improved Tissue/Cell RNA kit (Spark Jade: AC0202) in the light of the manufacturer's recommended procedures. Briefly, we added lysis solution to the treated NP cells in each groups, which were centrifuged at 13400 × g for 60 seconds. Next, an equivalent volume of 70% ethanol was added, and the solution was immediately purified over an RA adsorption column. Afterward, we added 700 μl deproteinized solution RW1 to the RW absorption tube. Then, the procedure which the RW rinsing solution was added and centrifuged was repeated twice. The RA adsorption column was placed back into an empty collecting tube. Finally, total RNA was extracted by adding sterile enzyme-free water to RNA adsorption columns and centrifugation. After the RNA concentration was determined by spectrophotometry, they are used to obtain cDNA with an SPARK script IIRT Plus Kit (Spark Jade: AG0304). The qPCR reaction which was mixed with 2 × SYBR Green qPCR Mix (Spark Jade: AH0104) was set at 94°C for 3 minutes. After the incubation at 94°C for 10 s and 60°C for 30 s. The relative expression levels of target genes and reference genes were quantified by 2^−ΔΔCT method. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the purpose of an internal control. The primers used are listed in Table 2.

The expressions of immune-related genes including TLR3, AjToll, MyD88, TRAF6, p50, p105, rel, MKK36 and p38 in the intestine of A. japonicus collected on day 40 were analyzed. AjToll is one of the Toll-like receptor genes identified from sea cucumber A. japonicus, which is functionally involved in the immune responses of A. japonicus [11 (link)]. Total RNA from the intestine was extracted using a SPARK easy Improved Tissue/Cell RNA Kit (SparkJade, Jinan, China). The cDNA was generated by Prime Script™ RT reagent Kit (Takara, Japan). The genes were determined using qPCR performed with SYBR^®® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primers were listed in Supplementary Table S1. qPCR was run in triplicate with the reference gene using the following protocols: 30 s at 95 °C, followed by 39 cycles of 5 s at 95 °C and 30 s at 57 °C. Data were quantified using the 2^−ΔΔCT method.