Taqman advanced mirna cdna synthesis kit

Reverse transcription was performed on the mRNA using the EvoScript Reverse Transcriptase (Roche, 07912323001, Mannheim, Germany) and Primer Random (Sigma-Aldrich, 11034731001, Mannheim, Germany). The reaction was carried out at 45 °C for 15 min and 85 °C for 5 min, and stopped by 65 °C for 15 min. Processed complementary DNA was stored at −20 °C. For gene expression, quantitative PCR was performed using Universal Probe Library (UPL Roche, Mannheim, Germany) system by Roche LightCycler480. ACTB was used as housekeeping gene. Primers and probes are shown in Table 3.
For miRNA, reverse transcription was performed using the “TaqMan^® Advanced miRNA cDNA Synthesis Kit” (Themofisher, A28007, Carlsbad, CA, USA) according to the supplier’s protocol. 10 ng total RNA plus 10 pM ath-miR159a as spike-in control were used per reaction. Processed cDNA was stored at −20 °C. Potentially relevant miRNAs were determined by a comprehensive survey of the current literature applying the PubMed interface. There were 18 miRNAs related to EMT, chemotherapy, and stemness in various types of cancer selected (Table 2). Quantitative PCR for expression of miRNA was performed using TaqMan^® Advanced MicroRNA Assay (Thermofisher, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Total RNA was extracted using TRIzol^® (Life Technologies) and purified with the RNAeasy mini kit (Qiagen). Universal reverse transcription (RT) was carried out with TaqMan™ Advanced miRNA cDNA Synthesis Kit (Termo Fisher Scientific) according to the manufacturer’s instructions. TaqMan MicroRNA assays for hsa-miR-146a (478399 mir) and TaqMan Fast Advanced Master mix were used for detection of miR-146a expression level carried out on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) under the following conditions: incubation for 20 s at 95 °C, followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Each sample was amplified in triplicate. miR-let7a and miR-361 were used as endogenous control.

10 ng of input RNA was used for cDNA synthesis using TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Carlsbad, CA, USA), miR abundance were measured by RT-PCR on a QuantStudio 6 (Thermo Fisher Scientific, Carlsbad, CA, USA) using Applied Biosystems Fast Advanced Master Mix (Thermo Fisher Scientific, Carlsbad, CA, USA).
Target miRNAs were miR-15a-5p, -23a-5p, -23b-5p, -499a-3p, -206, -208a-3p, 208b-3p, -451a, -486-5p, -126-3p, -1-3p, -133a-3p, -133b, -148b-3p, -30b-3p, -145-5p, and -16-5p Thermo Fisher Scientific, Cat# A25576, Carlsbad, CA, USA) (Supplementary Table 1). The geometric mean of three reference miRNAs (miR-361-5p, -320a, -186-5p; Vandesompele et al., 2002 (link)) was used for normalization based on miRNAs that showed the least variation amongst the participants between and within groups. The abundance of miRs were measured using the 2^(−ΔCT) method (Schmittgen and Livak, 2008 (link)).

Total RNA was quantified by density measurement after separation by agarose gel electrophoresis with ethidium bromide staining. Equal amounts of RNA were reverse-transcribed using the TaqMan advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific). Differential expression of miR-16 was validated in triplicate by using TaqMan miRNA advanced assays (Thermo Fisher Scientific). Digital PCR was performed on the QuantStudio 3D Digital PCR System using the GeneAmp PCR System 9700 (Thermo Fisher Scientific). After PCR, the chips were imaged on the QuantStudio 3D Instrument, which assesses the raw data and calculates the concentration of the cDNA sequence targeted by FAM- and VIC-labeled probes by Poisson distribution (65 (link)). For more in-depth analysis, the QuantStudio 3D AnalysisSuite was used to report the data as copies per microliter. The probe sequence used was miR-16–5p (5′-UAGCAGCACGUAAAUAUUGGCG-3′).

Total RNA was extracted using a TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction and subsequently treated with RNase-free DNase I (Fermentas, San Diego, CA, USA). For detection of mRNA expressions, cDNA synthesis was performed from 1 µg total RNA using a SuperScript First-Standard Synthesis system for RT-qPCR (Invitrogen Life Technologies). Real-time PCR was performed by an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using Quanti-Tect SYBR Green PCR mixture (Qiagen, Hilden, Germany). The qPCR primers were: MCT1: forward: 5′-GTGGCTCAGCTCCGTATTGT-3, reverse: 5′-GAGCCGACCTAAAAGTGGTG-3′; LDHA: forward: 5′-TGGAGTGGAATGAATGTTGC-3′, reverse: 5′-ATAGCCCAGGATGTGTAGCC-3′; and β-actin: forward: 5′-AGGCACCAGGGCGTGAT-3′, reverse: 5′-GCCCACATAGGAATCCTTCTGAC-3′. For detection of miRNAs, reverse transcription reaction was performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the instructions. qPCR was performed by ABI PRISM 7900 Sequence Detection System. The reactions were incubated in a 96-well optical plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. RNU6 was used as internal control. Relative expressions were calculated by the 2^−△△CT method. All reactions were performed in triplicate.