Sybr green master mix

The enrichment of H3.3A-GFP or ORC at different Drosophila Amplicon in Follicle Cells (DAFCs) was determined by qPCR. Forty cycles of a two-step PCR protocol (denaturation at 95° for 15 sec, and annealing/extension at 62° for 30 sec) were run. The analysis was done on a Stratagene (Santa Clara, CA) Mx3005P machine with SYBR Green Master Mix (600843; Agilent, Santa Clara, CA). For qPCR quantification, the amount of DNA in the pellet as expressed as percentage of input DNA estimated by a standard curve generated from a serial dilution of the input. The values were then normalized to two control, nonorigin, loci at cytogenetic position 64A and 93E/F or to 93E/F alone (Figure 4). Enrichment was also determined at the Drosophila hsp70 locus, as a positive control (Schwartz and Ahmad 2005 (link)).

The qPCR analyses were performed to see the changes in the expression profile of key genes of phenylpropanoid cascade and MAPK and WRKY transcription factors along with the heteromeric G protein, Gα, and Gβ in the wheat genotypes treated with B. sorokiniana, crude toxins, and different doses of Bipolaroxin at 7 days of inoculation under glasshouse conditions. Total RNA was extracted using RNA isolation kit (Agilent, Santa Clara, CA, USA) and cDNA was synthesized using cDNA synthesis kit (BioRAD, Hongkong, China) following the manufacturers’ instructions. The quality and quantity of cDNA were analyzed using a nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Expression analyses were performed using gene-specific primers (Supplementary Tables S2–S4) using sybr green master mix (Agilent, India) on the BioRAD Real Time PCR System (Model: MJ MiniOpticon, BioRAD, India) according to Singh et al. [42 (link)]. Actin was taken as the internal control. The relative transcript levels were calculated using the 2^−ΔΔC_T method [43 (link)].

Adipose tissue was finely minced, and samples were allowed to recover for 12 hours in a 37°C 5% CO₂ incubator in M199 media with 1nM of insulin and 40nM of dexamethasone as previously described for adipose tissue cell culture.40 mRNA was isolated from adipose tissue using the Omega Bio‐tek E.Z.N.A. Total RNA Kit II. The purity of RNA was confirmed by spectrophotometric analysis, and the quality assessed by Agilent 2100 Bioanalyzer. cDNA was generated by a reverse transcriptase reaction using qScript cDNA Supermix kit. Human primers were obtained for IL‐6, IL‐10, IL‐1β, and TNF‐α (RT² qPCR Primer Assay, QIAGEN). Cytokine gene expression was assessed by real‐time quantitative polymerase chain reaction (PCR) using the Biorad CFX Connect Real Time System and SYBRGreen Master Mix (Agilent). PCRs were performed in triplicate in 96‐well plates, The data were analysed with the ddCt method as previously described,41 after normalization to an internal control, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, zeta (YWHAZ), a routinely used housekeeping gene for studies in adipose tissue.42 Levels of the YWHAZ transcript did not change after the intervention. The results were expressed as fold change for each target gene from baseline. Real‐time PCR analyses were performed using 10 paired patients before and after surgery.