Universal cdna synthesis kit

Inhibition of miR-21 by LNA-anti-miR-21 was determined using RT microRNA real-time PCR. Nine days after the first LNA-anti-miR-21 injection, total RNA extraction was performed using GeneJET RNA purification kit (Thermo Scientific, Lithuania) according to the instructions. RNA concentration was adjusted to 5 nm. To synthesize cDNA from miRNAs, a universal cDNA synthesis kit was used (Exiqon, Denmark). Also, to enhance cDNA from miRNA and detect the amount of miR-21, a BIOFACT™ 2X real-time PCR master mix (BioFACT ™, High ROX, Korea), along with miR-21-5P and miR-16-5p specific primers, was used ( Exiqon, Copenhagen, Denmark) as internal control to normalize real-time PCR. We also used a StepOnePlus PCR system (Applied Biosystems, USA) for 40-cycle real-time PCR experiments, while 2^−ΔΔCT method was employed to calculate the relative miRNA-21.

Plasma samples were acquired by standard venipuncture and centrifugation in EDTA-coated vacutainer tubes (Becton Dickinson) and frozen at -80 °C until use. RNA was extracted from plasma using the mirVana PARIS Isolation Kit (Life Technologies), according to the manufacturer’s instructions with one modification; prior to the addition of acid-phenol:chloroform, 150Amoles of c-elegans miR 39 (Life Technologies) was added as an internal standard and RNA carrier. RNA was extracted from 100 μl plasma and eluted in 100 μl nuclease free water. A 4 μl aliquot of RNA elute was reverse transcribed in 20 μl reactions using the Universal cDNA Synthesis Kit (Exiqon), according to the manufacturer’s instructions.

For miRNA isolation, an RNA isolation kit (Exiqon) was used and complementary DNA was generated using a universal cDNA synthesis kit (Exiqon). PCR with reverse transcription (RT–PCR) was conducted using an ExiLENT SYBR Green Master Mix kit and LNA primer sets for miR-371-3p (cat #204299) as per the manufacturer's instructions. Absolute quantification of miR-371-3p was performed via standard curve method using synthetic oligonucleotides for miR-371-3p from Exiqon. RT–PCR was performed using an ABI Prism 7500 Real-Time PCR System as per the manufacturer's instructions.
For mRNA isolation, an RNAeasy kit (Qiagen) was used and RT–PCR was performed using Taqman RNA-to-C_T kit (Applied Biosystems) and Taqman probes from Applied Biosystems for PRDX6 (Hs00705355_m1), PLCβ4 (Hs00168656_m1), STX12 (Hs00295291_m1) and GAPDH (Hs02758991_g1). RT–PCR was performed using an ABI Prism 7500 Real-Time PCR System. mRNA results were normalized to GAPDH and RQ was analysed using the ABI software.

Three miRNAs, viz. miRNA-106a-5p, miRNA-146a-5p and miRNA-19b-3p, were selected for the study based on miRNA profiling in PBMCs isolated from elderly and non-elderly asthma patients and healthy subjects. The profiling results and the outline of the study are briefly presented in the Additional file 1. From the miRNAs whose expression significantly differed in asthmatics and controls in the profiling study, we chose those that had been previously tested in serum and were related to the presence of asthma or its clinical features [15 (link), 20 (link)]. Additionally, although not shown as significant in the profiling results, miRNA-126a-5p was also included in the analysis, as many studies have reported it to have an important role in anti-inflammatory responses [20 (link)].
The miRNA was isolated from the serum using a miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer's protocol. Equal volumes of RNA were used to prepare complementary DNA (cDNA) with the universal cDNA Synthesis Kit (Exiqon). They were used to perform quantitative PCR (StepOne Plus; Applied Biosystems, Foster City, CA, US) with miR-93 as a reference gene. Serum expression levels of miRNA-106a-5p, miRNA-146a-5p, miRNA-19b-3p and miRNA-126a-5p were evaluated with real-time PCR. miRNA expression is presented as 2^−ΔCT.

The qRT-PCR was used to determine the expression of miRNA-183, -96, and -182 in the hBMSCs after transfection. The RNA extraction was then carried out using miRCURY RNA isolation kit (Exiqon, Denmark) for 24 and 48 hours following cell transfection. Universal cDNA synthesis kit (Exiqon, Denmark) was similarly used for the synthesis of cDNA. The qRT-PCR was also performed via SYBR green master mix kit (Exiqon, Denmark), and specific primers were determined for miRNA-183, -96, and -182 (Exiqon, Denmark) Table 1. In addition, snord was employed as the internal control and the 2^−ΔΔCt method was used for data analysis in the qRT-PCR test. The qRT-PCR was then performed using the Rotor-Gene 6000 (Corbett, Australia) with the following thermal cycling profile: 95°C for 2 minutes, followed by 40 cycles of amplification (95°C for 5 seconds, 60°C for 30 seconds).