Cfx 96 real time pcr system detector

MiRNA expression was quantified by determining the cycle threshold (C_T) which is the number of PCR cycles required for the fluorescence to exceed a value significantly higher than the background fluorescence, using the TaqMan small RNA assay (Applied Biosystems by Life Technologies) with miRNA specific primers according to manufacturer’s instructions. Briefly, 1.4 μL of cDNA was added to 10 μL of probe qPCR mix and 7.6 μL of nuclease free water. The following TaqMan small RNA assay (Applied Biosystems) primers were used: hsa-miR-9-5p, hsa-miR-21-5p, hsa-miR-155-5p, and RNU6B. All analyzed miRNAs are of human (Homo sapiens) origin and therefore, the prefix “hsa” is omitted throughout the text. RT-qPCR reactions were performed using a CFX96 Real-Time PCR System Detector (Bio-Rad, Hercules, CA, USA). Samples were run in duplicate for each experiment. Data were analyzed using the comparative Ct (2-ΔΔC_T) method using the small nuclear RNA, RNU6B, as an endogenous control. To monitor reagent contamination, negative controls were included for each primer pair. PCR cycling conditions were as follows: 95 °C for 3 min 40 cycles of 95 °C for 15 s and 60 °C for 60 s.

Real-time PCR was carried out as described previously^{38 (link),39 (link)}. Total RNA was purified from brain tissue using TRIzol reagent (15596018, Invitrogen Life Technologies, Carlsbad, CA, USA) and was treated with DNase I (M610A, Promega, MO, USA) to eliminate genomic DNA contamination. Reverse transcription of total RNA (2 μg) was conducted in a volume of 20 μl using a reverse transcription system (A3500, Promega, MO, USA). Real-time PCR was performed with 10 μl of 2X iQ™ SYBR Green Supermix (#170882, Bio-Rad Laboratories, Foster City, CA, USA), 1 μl each of 5 pmol/μl forward and reverse primers, and 4 μl of complementary DNA (cDNA) (1/8 dilution of the conversion) in a total volume of 20 μl using a CFX 96 Real-Time PCR System Detector (Bio-Rad Laboratories). The Bio-Rad CFX Manager 3.1 was used to analyze qPCR data. The primer sets used in PCR were listed in Supplementary Data 5.

To analyze the expression of DE miRNAs (miR-16-5p, miR-199-3p (detection of both miR-199a-3p and miR-199b-3p), miR-374c-5p, miR-6886-3p, and miR-6856-3p), blood samples after MTB-specific peptide stimulation were treated using 500 µL of RNA/DNA stabilization reagent for blood/bone marrow (Roche Diagnostics, Mannheim, Germany). Subsequently, a MagNA Pure LC RNA Isolation Kit-High Performance kit (Roche Diagnostics) was used to extract RNA according to manufacturer’s instructions [30 (link)].
Next, complementary DNA (cDNA) of miR-16-5p, miR-199a-3p, miR-199b-3p, miR-374c-5p, miR-6886-3p, and miR-6856-3p was synthesized using the miRCURY LNA RT Kit (Qiagen) following manufacturer’s protocols. The temperature profile for cDNA synthesis reaction was as follows: 42 °C for 60 min and 95 °C for 5 min.
Quantitative reverse transcriptase (qRT)-PCR was performed using the miRCURY LNA miRNA PCR Assay [31 (link)]. PCR cycling was performed as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 56 °C for 1 min. qRT-PCR were performed on the CFX96 Real-time PCR System Detector (Bio-Rad, Hercules, CA, USA). Samples were run in duplicate for each experiment. To monitor contamination of the reagents, a negative control was included for each primer pair. Data were analyzed using the comparative ΔC_T method (2^−ΔCT), with RNU44 as an endogenous control [32 (link)].