Mysticq microrna sybr green qpcr readymix

TRIzol reagent (Thermo Fisher Scientific, Inc.) was used for total RNA extractions from tissue specimens and in vitro cultivated cells. Following reverse transcription performed using Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit, SYBR^® Green Quantitative RT-qPCR Kit (Sigma–Aldrich) was used to prepare qPCR mixture with 18S rRNA as endogenous control to detect the expression of lncRNA CASC11.
mirVana miRNA Isolation kit (Thermo Fisher Scientific, Inc.) was used for miRNA extractions from tissue specimens and in vitro cultivated cells. After reverse transcription performed with TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific), PCR reaction mixtures were prepared using MystiCq^® microRNA^® SYBR^® Green qPCR ReadyMix™ (Sigma–Aldrich, MO, U.S.A.) to detect miR-188-5p with U6 as endogenous control.
Each experiment included three biological replicates and data normalization was performed using 2^−ΔΔCq method.

Samples of RNA isolated from blood serum exosomes and PPM urine exosomes from CP/CPPS patients (n = 20 of 25 in total) and healthy men (n = 20 of 25 in total) were analyzed for select microRNAs known to be upregulated in PCa: hsa‐miR‐141, hsa‐miR‐375, hsa‐miR‐501, and hsa‐miR‐532. Reverse transcription (RT) of 100 ng of RNA into cDNA was achieved using the MystiCq® microRNA cDNA Synthesis Mix Kit (Sigma‐Aldrich, Taufkirchen, Germany; cat. no. MIRRT‐100RXN) according to the manufacturer's protocol. Quantitative PCR (qPCR) was performed using 20 ng cDNA per reaction, commercial primer sets for targeted microRNAs (Table S2), and MystiCq® microRNA® SYBR® Green qPCR ReadyMix™ (Sigma‐Aldrich; cat. no. MIRRM00‐100RXN) on the CFX96 Touch™ RealTime PCR Detection System (Bio‐Rad). SNORD44 (small nucleolar RNA, C/D Box 44) was utilized as a reference gene in the quantification of exosome microRNAs and was amplified using the Human Positive Control Primer (Sigma‐Aldrich; part of MIRRT‐100RXN). Quantitative PCR was performed in duplicate. Relative expression levels of microRNAs were calculated by the 2−ΔΔCT method. The Mann–Whitney U test was used to compare the relative microRNA levels in two groups, and P‐values < 0.05 were considered significant.

For validation by qPCR, we selected two miRNA genes that showed the highest fold change and the lowest p value in differential gene expression analysis in miRNA-seq between sensitized and control rats in adipose tissue and lung tissue (10 asthmatic and 13 control rats). The expression of the same miRNA genes was also examined in BALF-derived exosomes from these animals (n = 23). Exosomes from BALF were precipitated using miRCURY Exosome Isolation Kit (Qiagen, Wroclaw, Poland), and RNA and microRNA from BALF-derived exosomes were extracted using RNeasy mini kit (Qiagen). For reverse transcription, we used MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich, Darmstadt, Germany. Quantitative PCR was done using MystiCq microRNA SYBR Green qPCR ReadyMix (Sigma-Aldrich, Darmstadt, Germany) and microRNA assays for miR-151-5p, miR-423-3p, and RNU-6 (endogenous control). Differential expression results from qPCR were compared using Data Assist software (ThermoFisher Scientific, freely available form website) using the relative quantification method.