Nanodrop spectrophotometer

TRIzol® Reagent was used to extract the total RNA from the samples. The concentration and purity of RNA were also determined with a NanoDrop spectrophotometer (DeNovix, USA), the results were verified by agarose gel electrophoresis, and then the extracted RNA was immediately used in the next step. Based on the manufacturer’s instructions provided by the Parstous Easy cDNA Synthesis Kit (Parstous, Iran), cDNA was synthesized for both BAMBI and RBX1 genes.

The gastrointestinal regions of Drosophila melanogaster larvae were extracted from all experimental groups after 48 h of treatment. This was achieved by submerging third instar larvae in Poels’ salt solution (PSS). Subsequently, in accordance with previous methods [20 (link)], the extracted tissue was transferred to Eppendorf containers that were filled with TRIzol Reagent by using an RNA extraction kit (Bioserve RNA Extraction Kit, Hyderabad, India) to facilitate total RNA extraction. To determine the concentration and purity of the isolated RNA, the absorbance ratios at 260/280 and 230/260 nm were measured with a NanoDrop spectrophotometer (Denovix, Bioserve, Hyderabad, India).

Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific) and Phenol equilibrated, stabilized chloroform:isoamyl alcohol 25:24:1 (PanReac Applichem, Barcelona, Spain). A DeNovix Nanodrop Spectrophotometer was used to measure the RNA concentration. A total of 500 ng of total RNA was transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen) at 55°C. RT-qPCR was performed in a the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with β-actin as the reference gene under the following thermal cycling conditions: denaturation at 95°C for 30 s followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 30 s. Sequences of specific primers are listed in Table S3. Gene expression levels were normalized to those in SCR-treated mice.

EDTA anticoagulated blood samples (8 mL) were collected from eligible participants visiting the HIV Clinic at Satun Hospital, Satun Province every six months for follow-up testing and to receive antiretroviral therapy (ART). To separate the plasma and buffy coat, the whole blood sample was centrifuged at 900 × g for 10 min and then kept at –20 °C until further DNA extraction.
A total culture of 10⁷ promastigotes of L. siamensis (MON-324; MHOM/TH/2010/TR), L. martiniquensis (MON-229; WHOM/TH/2011/PG) and L. donovani (MHOM/ET/67/HU3) using Schneider’s Drosophila medium (Sigma, St. Louis, MO, USA) supplemented with 20% heat inactivated fetal bovine serum (GE Healthcare, Chicago, IL, USA) at 26 °C were harvested and washed three times with phosphate buffered saline (PBS) before DNA extraction.
The buffy coat was extracted for DNA using the Geneaid™ DNA Isolation Kit (blood) (New Taipei, Taiwan), whereas genomic DNA of each species of Leishmania parasites was extracted using the DNeasy Extraction Kit (tissue) (Qiagen, Hilden, Germany) according to manufacturer protocols. The concentration and quality of the extracted DNA were analyzed by Nanodrop spectrophotometer (Denovix, Wilmington, DE, USA) at 260/280 and 260/230 ratios and kept at –20 °C until further use.