Primescript rt reagent kit with gdna eraser perfect real time

All herbs in QYSXD and the decomposed components were purchased from Tianyin Tianjiang Pharmaceutical Co., Ltd. (Jiangyin, Jiangsu Province, China) and conformed to the standards of the Chinese Pharmacopoeia. The decoction included 45 g of Astragalus membranaceus (Fisch.) Bunge; 30 g of Pseudostellaria heterophylla (Miq.) Pax; 15 g each of Rehmannia glutinosa (Gaertn.) Steud., Atractylodes macrocephala Koidz., Persicae semen, and Ligusticum chuanxiong Hort; and 20 g each of Euphorbia humifusa Willd. and Kummerowia striata (Thunb.) Schindl. DSS (MW 36,000–50,000 Da) was obtained from MP Biomedicals (Irvine, California, USA). LPS was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). A PrimeScriptRT Reagent Kit with gDNA Eraser (Perfect Real Time) and SYBR® Premix Ex Taq™ II were purchased from TaKaRa Bio Inc. TRIzol was obtained from Invitrogen Scientific Co., Ltd. (Waltham, Massachusetts, USA). Antibodies against Drp1, FIS1, caspase-1, light chain 3 (LC3), and β-actin were purchased from Servicebio Corp. (Wuhan, China). Enzyme-linked immunosorbent assay (ELISA) kits for RIP1, RIP3, NLRP3, IL-1β, and IL-18 were purchased from Sigma-Aldrich Corp. Dihydroethidium (DHE) for ROS analysis and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Servicebio Corp. (Wuhan, China).

Total RNA was isolated from various rice tissues using Trizol reagent (Tiangen, Beijing, China), and analyzed with a Nanodrop 1000 spectrophotometer (Thermo Scientific, UT) for assessment of quality and quantity. For RT-PCR, first-strand cDNA was reverse transcribed from total RNA with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa). Real-time RT-PCR was performed with iQ SYBR Green Supermix (Bio-rad), using the real-time PCR system (Bio-Rad C1000 CFX96). The rice actin gene was used as an internal control. Primers used to quantify the expression of OsTKPR1 are listed in Additional file 7: Table S4.

We extracted total RNA from the roots and leaves of cotton plants as well as from the leaves of GaRPL18-overexpressing A. thaliana and WT plants using the RNAprep Pure Plant Kit. The RNA was used to prepare cDNA with the PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time; Takara). The Gahistone3 (Cotton_A_11188) and ubiquitin10 (accession: At4g05320) genes were used as internal controls for cotton and A. thaliana, respectively. We designed all qRT-PCR primers with the Primer Premier 6.0 program (Table 1). Diluted cDNA was used as the template for the qRT-PCR, which was conducted with SYBR^® Premix Ex Taq™ (Tli RNaseH Plus; Takara) and an ABI 7900 qRT-PCR System (Applied Biosystems, CA, USA). The three-step method involved the following PCR conditions: 40 cycles of 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s. We analyzed the dissociation curves for each reaction and used the 2^−ΔΔCT method [44 (link)] to calculate the expression level of each target gene. All reactions were conducted with at least three biological replicates.