Sensifast sybr no rox qpcr kit

Prior to qPCR analysis, RNA was reverse transcribed into cDNA with a reverse transcription kit (Fluidigm, CA, USA) according to the manufacturer’s instructions. celA1 expression was measured using soFast EvaGreen supermix with the low ROX qPCR kit (Bio-Rad, CA, USA) and the CFX96 qPCR system (Bio-Rad). The primers used for celA1 were (forward) 5′-ACACTCCGCAGTCCTACT-3′ and (reverse) 5′-TAGAGCGTCAGAATCGGC-3′. The number of mycobacterial cells in the sample was quantified using the SensiFAST SYBR no-ROX qPCR kit (Bioline, London, UK) on bacterial DNA according to the manufacturer’s instructions. The primers used for Mmr quantification were targeted against the 16S-23S locus AB548718 (forward, 5′-CACCACGAGAAACACTCCAA-3′; reverse, 5′-CACCACGAGAAACACTCCAA-3′). Each bacterial quantification qPCR run included a standard curve of the known amounts of Mmr DNA. The mycobacterial cell number in each sample was used to normalize celA1 expression.

For RT–qPCR analysis, RNA was isolated with TRIzol (Thermo Fisher Scientific), treated with RQ1 DNase I and reverse-transcribed using the Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific). Ago2, Hbs1l and Gapdh were quantified using sensiFAST SYBR No ROX qPCR kit (Bioline). Homer and Snord15b were used to estimate the efficiency of soma/neurite separation. See Supplementary Table 7 for primer sequences. Relative expression levels were calculated using the ΔΔC_t method with Gapdh as a reference gene.

RNA from soma and neurites was treated with RQ1 DNase I to remove traces of gDNA and reverse-transcribed using the Maxima first strand cDNA synthesis kit (Thermo Fisher). qPCR was performed with sensiFAST SYBR No ROX qPCR kit (Bioline) with the primers provided in Supplementary Table S1. RT-qPCR reactions were carried out in technical duplicates and biological triplicates (soma) or duplicates (neurites), using a CFX96 Real-Time PCR system (Biorad). Relative neurites/soma expression levels were calculated using ΔΔC_t method, with Capdh as a reference RNA.

RNA from soma and neurites was treated with RQ1 DNase I, reverse-transcribed using the Maxima first strand complementary DNA synthesis kit (Thermo Fisher), and quantified by quantitative PCR (qPCR) using sensiFAST SYBR No ROX qPCR kit (Bioline). The following primers were used (PrimerBank ID): Nxf7 (13561071a1), Vangl1 (29164511a1), ldlrap1 (160333774c1), Col3a1 (20380522a1), Crtap (225543172c1), Tagln (291045204c1), Bmper (24371215c1), Lamb1 (114326496c1), Myo1c (124494243c3), Mme (31543255a1), Stx3 (924268a1), Nid2 (26343027a1), Mbnl2 (140971799c1), St3gal6 (118130739c1), Mov10 (254540178c1), H3f3a (6680159a1), H2afy2 (133892300c1), Gng3 (6754022a1), Fbll1 (148539881c1), Nup210 (172073151c1), Tubb3 (12963615a1), rRNA (fw: 5′-aaacggctaccacatccaag-3′, rev: 5′-cctccaatggatcctcgtta-3′). Relative neurites/soma expression levels were calculated using ΔΔC_t method, with rRNA as a reference RNA.

Total RNA from the early and late passages were extracted with a Zymo Quick-RNA MiniPrep kit (R1054, Zymo Research, Tustin, CA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were evaluated with a NanoDrop One spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The SensiFAST cDNA synthesis kit (BIO-65053, SensiFAST, Bioline, Memphis, TN, USA) was used to synthesize cDNA from RNA. Each PCR reaction was conducted in a 20 μL reaction mixture with a final amount of 10 ng cDNA for each mixture using the SensiFAST SYBR No-ROX qPCR kit (BIO-98005, Bioline, Memphis, TN, USA). Quantitative RT-qPCR experiments were carried out with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA). The cycling parameters were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, an optimized annealing temperature for 10 s, and 72 °C for 5 s. The results of each target gene were normalized to β-actin mRNA expressions. The primer sets were designed using the Primer3 online software (https://primer3.ut.ee/) (accessed on 21 November 2021). The list of primers used for the experiments is shown in Table S1. Each experiment had three replicates per condition, and a statistical analysis was performed with a Mann–Whitney t-test using GraphPad Prism 9.3.1.