Dynamo hs sybr green

RNA was isolated using the GeneJET RNA isolation kit (Thermo Fisher Scientific #K0732) according to the manufacturer’s instructions. Genomic DNA was digested using an on-column DNase step (Sigma-Aldrich #04716728001) for 15 min. RNA was converted into cDNA using the High Capacity cDNA Reverse Transcriptase kit (Thermo Fisher Scientific #43-688-13) according to the manufacturer’s instructions. cDNA was synthesised according to the following steps: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min.
RT-qPCR was performed by using the Brilliant III SYBR® Green QPCR Master Mix (Agilent #600882) or DyNAmo HS SYBR Green (Thermo Scientific #F410L) and the QuantStudio 3. The following RT-qPCR cycling parameters were used: initial denaturation on 95 °C for 10 min, 40 cycles of 95 °C for 20 s, 57 °C for 30 s and 72 °C for 30 s, finished by a dissociation step 65–95 °C (0.5 °C/s). Samples were run in technical triplicates. Fold change expression was determined using the 2^−ΔΔCT method.

Larvae (5 dpf, n=30) were pooled and total RNA was isolated using RNeasy mini kit (cat# 74104, Qiagen) and reverse transcriptase reaction was performed by using Superscript III (cat# 18080093, Thermo Fisher Scientific) according to the manufacturers' protocols. Quantitative Real-Time PCR (qPCR) reaction (primers: Table S1) was undertaken with DyNAmo HS SYBR green (F410L, Thermo Fisher Scientific) cycling (40 times) at 95°C 25 s, 57.5°C 30 s, and 70°C 45 s followed by a standard melt curve (QuantStudio3, Applied Biosystems).

Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Retinas from vehicle-injected mice and flibanserin-injected mice were harvested 48 hours and 72 hours after light exposure. Total RNA was extracted from retinas using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was converted to cDNA using a Bio-Rad iScript cDNA synthesis kit (Hercules, CA). Primers were designed with the Integrated DNA Technologies Realtime PCR Tool (S1 Table, Coralville, Iowa). PCR reaction mixtures consisted of 10 μL DyNAmo HS SYBR green (Thermo Scientific, Waltham, MA), 1 μL cDNA template, 1 μL of 200 μM gene-specific primer sets, and 8 μL distilled water. Using a Bio-Rad Chromo4 System (Hercules, CA), samples were initially denatured for 2 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 58°C for 30 seconds. Gene expression was normalized to β-actin and relative gene expression in flibanserin-injected mice was compared to vehicle-injected mice using the ΔΔCt method.

Total RNA was isolated from ventral bone and cartilage of juvenile golgb1^Q2948X and golgb1^X3078 genotyped fish (60 and 63 dpf, respectively, n=3 per genotype) using RNeasy mini kit (74104, Qiagen). Subsequently, a reverse transcriptase reaction was performed by using Superscript IV (18091050, Thermo Fisher). Zebrafish galnt3 (XM_009300463.2) coding sequence was confirmed by multi-species nucleotide BLAST (NCBI) leading to galnt3 forward, 5′-TCCTTCAGAGTGTGGCAGTG and reverse, 5′-TGATGGTGTTGTGGCCTTTA primers. gapdh was used as a reference gene (forward, 5′-TGTTCCAGTACGACTCCACC and reverse, 3′-GCCATACCAGTAAGCTTGCC). Quantitative real-time PCR (qPCR) reactions (quadruplicates per individual) using DyNAmo HS SYBR green (F410L, Thermo Fisher) with PCR cycles (40 times) of 95°C for 25 s, 57.5°C for 30 s and 70°C for 45 s, followed by a standard melt curve were applied (QuantStudio3, Applied Biosystems).

MiRNA expression profiling of leukemic cells from six paired initial diagnosis-relapse samples AML cases were performed. MiRNA expression was measured on Taqman Low Density Arrays (TLDA) v2.0 using Taqman technology in the 7900 HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
Differentially expressed miRNAs were validated by single stem loop real-time PCR using TaqMan MiRNA assay (Applied Biosystem) according to the manufacturers' protocol. The expression levels of miRNA target genes (BIM and p21^WAF1/CIP) that had been identified in other types of cancer, were validated with RT-qPCR in 14 paired initial diagnostic-relapse MLL-rearranged AML samples, six pairs in the discovery cohort and eight additional paired samples with MLL-rearrangements (9 AF10, 2 AF9, 1 ELL, 1 ENL, and 1 unknown) in the validation cohort. Primers are shown in Supplementary Table S4. Messenger-RNA expression was performed by RT-qPCR using DyNAmo HS SYBR green (Thermo Scientific, Waltham, MA, USA). The average cycle threshold (Ct) value was used to calculate miRNA and mRNA expression levels relative to the expression level of the reference RNU24 or GAPDH, respectively, using the comparative Ct method [36 (link)].