Taqman mirna primers

miRNA expression was evaluated on a total of 75 SCC tissues, 5 poorly differentiated NET and 5 differentiated NET compared to the respective normal adjacent tissues.
cDNA synthesis was performed using a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) for three miRNA candidates (miR-133b, miR-449a, and miR-223) in accordance with the manufacturers’ instructions. The expression levels of miRNA candidates were detected using TaqMan Fast universal PCR master mix (Applied Biosystems, CA, USA) via the ViiA 7 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with each primer for TaqMan miRNA primers (Applied Biosystems, Foster City, CA, USA).
Real-time PCR was performed on a ViiA 7 real-time PCR system. The Ct value of each miRNA was determined using ViiA 7 software (Applied Biosystems, Foster City, CA, USA) and setting a threshold of 0.2 Ct values. For miRNAs undetermined by the instrument the threshold was set up at 35.0. For calculating the ∆Ct of miRNAs of interest, Ct values of each miRNA were normalized with U6 small nuclear RNA (snRNA) as an endogenous control, and mean Ct values of a miRNA across five pools were used. The relative miRNA expression was calculated with the ∆∆Ct method. FC was calculated using the 2 ^−∆∆Ct method [10 (link)]. Each sample was run in triplicate.

qRT-PCR was performed using TaqMan miRNA primers and RT-PCR kit (Life Technologies) on a CFX Connect Real-time System (Bio-Rad, Hercules, CA, United States) with snRNA U6 as an endogenous control as described before.

Total RNA was prepared using the miRVana miRNA isolation kit (Life Technologies, Foster City, CA, USA) or the RNeasy (Qiagen, Frederick, MD, USA) for miRNA or mRNA studies, respectively, as described previously^{50 (link),86 (link),87 (link)}. Quantitative (q)RT-PCR for miRNAs was performed using Taqman miRNA primers and RT-PCR kit (Life Technologies) with snRNA U6 as an endogenous control as described before^{50 (link),87 (link)}. For protein-coding genes, qRT-PCR was performed using a QuantiFast SYBR Green RT-PCR kit and QuantiTect primers (Qiagen) with 18 s rRNA as endogenous controls^{50 (link),51 (link),87 (link)}.

Reverse transcription of whole embryo lysate samples was performed using a MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, P/N: 4366596) in addition to separately ordered TaqMan miRNA primers (Thermo Fisher Scientific, P/N: 4427975) for each of the 6 targeted miRNAs and snU6 according to the manufacturer’s instructions: 10 µL of RT Mastermix, 1 µL of whole embryo lysate and 4 µL of water were added to each RT reaction before thermal cycling at 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min in both cases. For qPCR amplification of whole embryo lysate samples, 1.33 µL of RT reaction products was mixed with 18.67 µL of TaqMan 2X Universal PCR Mastermix No AmpErase UNG (Thermo Fisher Scientific, P/N: 4324018), and quantitative amplification was performed using a CFX384 thermocycler (BioRad, Mississauga, ON, Canada) for 40 cycles.

A miRNA panel consisting of five miRNAs (miR-16, miR-29b, miR-126, miR-155, miR-200c) involved in angiogenesis and PE pathogenesis was quantified in plasma samples using RT-qPCR. Briefly, whole blood was collected in EDTA tubes. Samples were centrifuged within 30 min after collection (1500×g, 15 min) at room temperature and stored at −80°C. Plasma samples were thawed on ice and centrifuged for 10 min (4°C, 16000×g). RNA enriched for small RNAs (including miRNAs) was isolated using the mirVana Paris Kit (Thermo Fisher). Reverse transcription and pre-amplification were performed using TaqMan miRNA primers (Thermo Fisher) and multiplex qPCR was done in a CFX96 thermal cycler (Bio-Rad) [25 (link)]. Raw C_q values were calculated in Bio-Rad CFX manager software v.3.1 using automatic baseline and threshold settings. MiRNAs with coefficient of variation values > 4%, indicating high technical variability, were excluded from the analysis. Data were normalized using spike-in Cel-miR-39 and relative miRNA levels were expressed as log(2^−ΔC_q * 10⁶).