Taqman microrna kit

The levels of miR-126, VEGFA, and Ets-2 mRNAs were measured in the HUVECs at 48 h after the treatments. Total RNA was extracted using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA was diluted and reverse-transcribed using the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA, USA). cDNA was prepared from 1 μg of RNA from each sample via RT-PCR with random primers or with miR-126 and U6 primers. The RT-PCR products were then amplified using the TaqMan microRNA kit (Applied Biosystems, Foster City, CA, USA).
The levels of VEGFA, Ets-2, and GAPDH mRNAs were determined using the SYBR Green Real-time PCR Quantitation Kit (Invitrogen, USA) using GAPDH as an internal reference. We used the same kit to determine the expression levels of miR-126 mRNA and U6 snRNA, using U6 as an internal reference. The PCR reaction conditions were as follows: denaturation at 94°C for 3 min, followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and synthesis at 72°C for 35 s. Fold changes in the expression levels of miR-126, VEGFA, and Ets-2 mRNA were calculated using △CT and 2^-△△CT. All assays were performed according to the manufacturer’s instructions.

In the ‘discovery set’, a global microRNA expression profiling was performed in lymph nodes from six FFPE oral tongue SCC samples (four with macrometastases and two with non-metastatic nodes) using TaqMan Human MicroRNA Arrays (Applied Biosystems, Foster City, CA, USA).
Total RNA (40 ng) from each lymph node sample were reverse transcribed into cDNA using the TaqMan microRNA Kit and Megaplex RT Primers (both from Applied Biosystems). After synthesis, the cDNA was pre-amplified using the TaqMan PreAmp Master Mix Kit and Megaplex PreAmp Primers (Applied Biosystems). The amplified-cDNA was then transferred to the TaqMan Human MicroRNA Array plates and the amplification was carried out in an Applied Biosystems 7900HT Real-Time PCR system.
The data obtained was analyzed using the software DataAssist v3.0 (Applied Biosystems). The fold-change difference between metastatic and non-metastatic lymph node samples was calculated using the 2^-ΔΔCt method [39 (link)]. The small nuclear RNA U6 was used as an endogenous control and the non-metastatic group was assigned as a reference since this was the most stable control in the assay.

Neurons from the CA1-CA3 regions of the hippocampus were collected by a PixCell IIe Laser Capture Microdissection Microscope (Life Technologies) from Naive, Severe TBI and Severe TBI + CM treated rats. Cells were lysed in 100μl of lysis buffer and total RNA was isolated using RNAqueous Micro Kit (Ambion). Total RNA was assessed for quality and concentration on an Agilent Bioanalyzer (Agilent Technologies) using the Pico Kit (Agilent Technologies). 1 ng of total RNA was reverse transcribed using the Taqman MicroRNA kit (Applied Biosystems). QPCR was performed on a Roche Light Cycler 96 using the microRNA Taqman probes to miR-212 and miR-9 (n = 3 biological replicates). Data analysis was performed using the ΔΔCT method with Severe TBI and Severe TBI +CM compared relative to naive levels.

Total RNA was extracted with TRIzol Reagent (Waltham, MA, USA), mRNA levels were quantified by use of the SYBR Green Premix Reagent (Takara Bio Inc., Shiga, Japan), and microRNA levels were detected by use of a TaqMan microRNA kit (Applied Biosystems, Foster City, CA, USA) following the manufacturers’ protocols. The endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was used for normalization.

According to the manufacturer’s protocol, total RNA was isolated from GC cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA). To detect mRNA, first-strand cDNA was synthesized by PrimeScript RT reagent kit (TaKaRa, Dalian, China). RT-qPCR was then performed using the ABI PRISM 7500 Fast Real–Time PCR System (Perkin Elmer/Applied Biosystems, Rotkreuz, Switzerland) with SYBR Premix Ex Taq™ II (Takara) according to the manufacturer’s instructions. U6 snRNA was used as an internal control to normalize the experimental results. As for miRNA detection, cDNA is reverse transcribed from total RNA samples using small RNA-specific, stem-loop RT primer from the TaqMan small RNA Assays and reagents from the TaqMan microRNA reverse transcription kit. MiR-195 levels were then detected using a TaqMan microRNA kit (Applied Biosystems) and normalized to U6 snRNA. The relative amount of miRNA was calculated using △△Ct. The Assay ID of miR-195 probe and U6 snRNA probe provided by ABI (USA) were 000494 and 001973, respectively. The sequences used in this work were listed as follows: AKT3 forward primer, 5’-TGAAGTGGCACACACTCTAACT-3’; AKT3 reverse primer, 5’-CCGCTCTCTCGACAAATGGA-3’; U6 RT primer, 5’-AAAATATGGAACGCTTCACGAATTTGG-3’; U6 forward primer, 5’-CTCGCTTCGGCAGCACATATACT-3’; U6 reverse primer, 5’-ACGCTTCACGAATTTGCGTGTC-3’. Each reaction was performed three times, independently.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. For mRNA detection, first-strand cDNA was synthesized using a PrimeScript RT reagent kit (Perfect Real Time; Takara, Dalian, China). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq™ II kit (Takara, Dalian, China) on a CFX96 real-time PCR system (Bio- Rad, Hercules, CA, USA). The PCR conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. β-Actin was used as an internal control to normalize the results. For miRNA detection, miR-221 levels were determined using a TaqMan microRNA kit (Applied Biosystems) and normalized to small nuclear RNA (Rnu6), which served as a control; the data were expressed as the log 2 fold change in respective miR/U6 snRNA levels. Primers for miR-221 and U6 reverse transcription and amplification were designed by and purchased from RiboBio Co., Ltd. (Guangzhou, China).

Total RNA was extracted using TRizol (Invitrogen), treated by DNase I (TaKaRa) for the removal of the genomic DNA, and then reversely transcribed into cDNA by reverse transcriptase M-MLV (TaKaRa). Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, USA) was employed to test the KLF4 mRNA expression, and the result was expressed in 2^-ΔΔCt. Primer sequences were as follows: KLF4-F: 5′-ATGGCTGTCAGCGACGCGCTGC-3′, KLF4-R: 5′-TTAAAAATGCCTCTTCATGTGTAAGGCG-3′; GAPDH-F: 5′-GCACCGTCAAGCTGAGAAC-3′, GAPDH-R: 5′-TGGTGAAGACGCCAGTGGA-3′.
RNAiso technology (TaKaRa, Dalian, China) was applied to isolate the total RNA from cancer cells and solid tumors. The expression of miR-152-3p was analyzed by a TaqMan RT kit (Applied Biosystems) and TaqMan MicroRNA kit (Applied Biosystems) under the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, USA), and 2^-ΔΔCt was performed to normalize the miR-152-3p expression. The primers were designed as follows: miR-152-3p-F: 5′-ACACTCCAGCTGGGTCAGTGCATGACAG-3′, miR-152-3p-R: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCAAGTT-3′; U6-F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, U6-R: 5′-CGCTTCAGAATTTGCGTGTCAT-3′.