Taqman primer probe mix

Total miR-1290 plasma levels were then quantified using the ddPCR system (Bio-Rad Laboratories, Hercules, CA). Briefly, 5 μl of the synthesized cDNA were added to a 20 μl PCR reaction mixture containing 10 μl of digital PCR™ supermix (Bio-Rad Laboratories), 1 μl of TaqMan primer/probe mix (Applied BioSystems) and RNase-free H₂O. Droplets were generated by loading the mixture into a plastic cartridge with 70 μl of QX100 Droplet Generation oil (Bio-Rad Laboratories). Cartridges were then placed into the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets generated from each sample were transferred to a 96-well PCR plate (Eppendorf, Hamburg, Germany), and PCR amplification was carried out on the C1000 Touch Thermal Cycler (Bio-Rad Laboratories), according to the manufacturer’s protocol. The plate was then loaded on the QX200 Droplet Reader (Bio-Rad Laboratories) and read automatically. The fraction of PCR-positive droplets was quantified assuming a Poisson distribution^{49 (link)}. In details, the QuantaSoft software was used to obtain the concentration results in no. copies per microliter (no. copies/μl) for each sample. Then, in order to convert these results in the no. copies present in the starting sample, the above concentration values were multiplied for the reaction volume, and divided for the starting volume of cDNA.

Following a previously established protocol, targeting the conserved REPL repeats specific for L. donovani and L. infantum, qPCR was performed with extracted template DNA from whole blood and DBS (S1 Table) [2 (link),17 (link)]. In brief, to prepare a 20 μL reaction mix, 9 μL template DNA, 10 μL of TaqMan Gene Expression Master Mix (Applied Biosystems) and 1 μL pre-ordered Taqman primer-probe mix (Applied Biosystems) were combined. For amplification, Bio-Rad CFX96 iCycler system was utilized. The following were the amplification conditions: 10 min at 95°C, followed by 15 seconds at 95°C and 1 min at 60°C for 45 cycles. In each run, a standard curve was created with 10 ng—1 fg of parasite DNA isolated from in vitro cultured promastigotes (L. donovani MHOM/IN/80/DD8) corresponding to 10,000–0.1 parasites per reaction. One reaction with nuclease-free water was also included in each run as a negative control. A sample was considered as negative with a cycle threshold (Ct) > 40. Each sample was evaluated in duplicate, and an additional run was made in case of an inconclusive result.

The allele frequency of the identified variant was validated using real time PCR by performing the allelic discrimination test using the same primers designed for Sanger sequencing as described above (Applied Biosystems). Briefly, PCR reactions were carried out using 25 µl reaction mixture containing 25 ng DNA, 1X Taqman primer/probe mix (Applied Biosystems), 1X genotyping Taqman master mix (Applied Biosystems) and sterile DNA/RNA free water. In each experiment, positive (cases for which genotype was confirmed by Sanger sequencing) and negative (water) controls were included. A batch of an ethnically matched Arab control DNA panel collected from 100 normal subjects that belong to different Arabian tribes was run in parallel. Genotyping steps were performed in a 7500 Fast Real-Time PCR System according to the manufacturer’s instruction (Applied Biosystems).

Quantitative Reverse Transcription PCR (RT-qPCR) for immunoglobulin heavy chain genes was performed at the Genomics Core Facility at AECOM, Bronx, NY, supported by the Cancer Center Support Grant (P30 CA013330). cDNA was prepared from 1 µg of total RNA using SuperScript™ VILO™ master mix (Invitrogen). RT-qPCR was performed using custom TaqMan assays designed for IGHG1-4 individual genes (Applied Biosystems) in 8 µL reaction volume containing TaqMan Fast Advanced Master Mix and TaqMan Primer Probe Mix as per manufacturer’s instructions and the 7900HT Fast Real-Time PCR instrument (supplementary Table 3). Eukaryotic 18S rRNA was used as the internal loading control (HS99999901_s1, Thermo Fisher Scientific). Fold-changes were calculated using the 2^−ΔΔCT method and presented as fold differences of relative gene expression normalized to human control RNA.

Brains were extracted from male and female WT and heterozygous Oprk1-Cre mice (age range, ∼8–12 weeks) and flash frozen. Brains were homogenized using a Dounce homogenizer, and total RNA was isolated using the RNAEasy Midi Kit (Qiagen) following manufacturer instructions. RNA was treated with DNase (Ambion). Total RNA was quantified on a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). RNA (1 mg) was reverse transcribed using the High-Capacity Reverse Transcription Kit from Applied Biosystems. cDNA was diluted 1:5, and 2 µl of that solution were subjected to real-time PCR amplification to detect Oprk1 (Mm01230885_m1, Thermo Fisher Scientific) with 5 µl of 1× SSoAdvanced Universal Probes Master Mix (BIO-RAD), 0.5 μl of 20× TaqMan Primer/Probe Mix (Applied Biosystems) in a total volume of 10 μl. Data were normalized to the endogenous control genes for transferrin (Tfrc; Mm00441941_m1, Thermo Fisher Scientific) or gusducin B (GusB; Mm019768_m1, Thermo Fisher Scientific).

Total RNA was isolated from cultured cells or tissues samples with Trizol reagent (Invitrogen, Carlsbad, CA, USA). MiRNAs were quantitated by real-time PCR using the TaqMan MicroRNA Assays (Invitrogen, USA) [18] . First-strand complementary DNA (cDNA) synthesis was carried out using 1 µg of total RNA in a 12 µl final volume containing 2 M stem-loop primer and 10 mMdNTP Mix (Invitrogen, USA). The mix was incubated at 65°C for 5 min, and then, 5xRT buffer, 0.1 M DTT, 200 U/µl MultiScribe reverse transcriptase and 40 U/µl RNase inhibitor were added (Invitrogen, USA). Then the mix was incubated at 37°C for 55 min, followed by 70°C for 15 min, and then held at −20°C. Real-time PCR was performed using a standard TaqMan PCR protocol [18] . The 20 µl PCR reactions included 1 µl of RT product, 1× Universal TaqMan Master Mix and 1× TaqMan probe/primer mix (Invitrogen, USA). All RT reactions, including the no-template controls, were run in triplicate. All mRNA quantification data were normalized to U6 expression. The relative amount of transcript was calculated using the comparative Ct method (Table S2).