Taqman mirna reverse transcriptase kit

Total RNA was extracted using the miRVana miRNA isolation kit (Ambion, #AM1560, Austin, TX, USA). RNA quality and concentrations were analyzed on a NanoDrop M-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. RNAs with quality scores >7.00 were used for expression assays. RNA concentrations were standardized to 200 ng/μL. TaqMan low-density miRNA arrays (TLDAs) (Applied Biosystems, #4444913, Foster City, CA, USA) were used to assess miRNA expression levels in proliferating basal cells grown in SAGM-EA. Reverse transcription of 600 ng total RNA was carried out using a TaqMan miRNA reverse transcriptase kit (Applied Biosystems, #4366596) with Megaplex RT primers, Human Pool (Applied Biosystems, #4399966). Samples were loaded onto the TLDA, which utilizes 384 wells preloaded with specific miRNA probes and primers in each well. The TLDA data were processed on an Applied Biosystems Model 7900 Genetic Analyzer, and the data were analyzed using the Applied Biosystems StatMiner software. Each sample was analyzed in triplicate, and each Ct value was normalized to the Ct value of RNU48 endogenous RNA control. Relative quantification of each miRNA was performed using the ΔΔCt method. Statistical significance of the fold change was assessed using two-tailed t-tests. p-values of <0.05 were taken as statistically significant.

Approximately 35 ng of total RNA was reverse-transcribed in a 10-uL volume using the TaqMan miRNA reverse-transcriptase kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Three microliters of the reverse-transcription reaction was used in each of the real-time PCR assays. Analyses of miR-122 were carried out in triplicates by means of the TaqMan human miRNA assays (Applied Biosystems) using SDS 7000 system software v1.2.3 (Applied Biosystems, USA).

Total RNA was obtained from exosome samples using the TRIzol reagent (Tiangen, Beijing, China) as described in the manufacturer's instructions. The quality and quantity of the extracted RNA were confirmed by the One Drop OD-1000 + Spectrophotometer (One Drop Technologies, Nanjing, China). TaqMan miRNA reverse transcriptase kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to synthesize cDNA with 200 ng total RNA as a template. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The qRT-PCR analysis was performed using TaqMan Universal Master Mix II no UNG (Thermo Fisher Scientific, Inc.) and commercial primers on the Applied Biosystems (Carlsbad, California, America) 7500 Real-Time PCR System. The experimental data was analyzed using the 2^−ΔΔCt method. All data are the average of three independent experiments.

RNA was isolated from P21 pellets of 0.5, 1.0, 1.5, 3.0, 4.5, 9.0, and 13.5 mL urine fractions that were resuspended in 0.1 mL of 1X PBS using miRNA serum/plasma kit (Qiagen). Cel-miR-39 was spiked in the qiazol solution as per manufacturer’s instructions. For the SN21^TCEP as well, the RNA was isolated using the same kit. RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific). Reverse transcription was performed using Taqman miRNA reverse transcriptase kit (Thermo Fisher Scientific). Primers for miR-16, miR-155, miR-200b, miR-203, and Cel-miR-39 were used for reverse transcription followed by real time QPCR (Quantstudio3, Thermo Fisher Scientific). The results were then exported and the Ct values for each of the miRNA were normalized against the spike-in control (Cel-miR-39) and the obtained dCt values for the urine P21 pellets from various fractions were compared.