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Transferrin saturation (TS) was measured using an iron and iron binding capacity kit (Sigma-Aldrich, Castle Hill, Australia).
Total RNA was digested with DNAse amplification grade (Invitrogen), one microgram was reverse transcribed using pd(N 6 and NotI d(T 18 primers and the High Capacity kit (Applied Biosystems), and the qPCR was carried out in 20 µl on an ABI Prism 5700 sequence detection system.
cDNA was generated using a High Capacity kit (Applied Biosystems, Foster City, CA, USA).
A total of 2 μg of RNA were reverse transcribed using a high capacity kit (Applied Biosystems, Rotkreuz Zug, Switzerland).
A 0.75- μg RNA aliquot of each RNA sample was reverse transcribed using a two-step PCR protocol (High Capacity Kit).
RNA was DNase-treated (Promega, Madison, WI, USA) and 2 mg of RNA were reverse transcribed using a high capacity kit (Applied Biosystems, Rotkreuz Zug, Switzerland).
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Crude RNA was repurified using a RNeasy Mini Kit (Qiagen, Hilden, Germany) followed by reverse transcription into cDNA using a high-capacity kit (Life technologies, Darmstadt, Germany).
Quantitative PCR was carried out using TaqMan® Gene Expression assays (Life Technologies): Hs01003267_m1 [hypoxanthine phosphoribosyltransferasesferase 1); Hs99999905_m1 [GAPDH (glyceraldehyde-3-phosphate dehydrogenase)]; and Hs00265315_m1 (KCNJ2), following reverse transcription with a high-capacity kit (catalogue number 4374966, Life Technologies).
RNA was purified using the RNeasy Mini or Plus Micro Kits (Qiagen), and reverse transcribed with the High Capacity RT kit (Applied Biosystems).
Assaying of the antioxidant activity was performed using the Total Antioxidant Capacity Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) using the 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic 2,2′-azino-bis 3-ethyl-benzothiazoline-6-sulphonic
Total RNA (10 ng/µL) was reverse transcribed using random hexamer primers with the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA).
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