Taqman microrna assay

RNA was extracted from cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). TaqMan™ MicroRNA Assay (Assay ID: 467534_mat) (Applied Biosystem, Foster City, CA) was used to measure miR-155 levels. cDNA was synthesized using miR-155 reverse transcriptase (RT) primers (TaqMan™ MicroRNA Assay), 10× RT buffer, 100 mM dNTPs in presence of RNase inhibitor (Applied Biosystem, Foster City, CA). Reverse transcription was done using the conditions (16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min) provided with TaqMan™ MicroRNA Assay in thermal cycler (T100™, Bio-Rad, Hercules, CA).
PCR was performed using miR-155 specific primers (TaqMan™ MicroRNA Assay) and TaqMan™ Universal Master Mix II, with UNG (Applied Biosystem, Foster City, CA) using the conditions specified in the assay (50 °C for 2 min, 95 °C for 10 min, (95 °C for 15 s, 60 °C for 60 s) ×40 cycles). U6 snRNA (TaqMan™ microRNA Control) assay was used as control for each sample under similar conditions as miR-155 assay. No template control was used for both control and miR-155 assay. Normalized results relative to control samples were reported.

The quantitative comparison of polymerase chain reaction (qPCR) products was performed by real-time PCR. qPCR of miRNA (reaction mixture: 10.0 μL of 2 × Universal PCR Master Mix II [Applied Biosystems], 7.0 μL of distilled water, 1.0 μL of TaqMan MicroRNA assay, and 2.0 μL of reverse transcription product) was done with a CFX96™ Real-Time System (Bio-Rad) (at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min). qPCR of mRNA was performed according to the method described previously [10 (link)]. Primers used were as follows: HSPA1B, F-TGGACTGTTG GGACTCAAGG AC, R-GGAACGAAAC ACCCTTACAG TATCA; HSPA8, F-TGCTGCTGCT ATTGCTTACG, R-TCAATAGTGA GGATTGACAC ATCA; p21, F-GACTCTCAGG GTCGAAAACG, R-GGATTAGGGC TTCCTCTTGG; and GAPDH, F-CACCACCATG GAGAAGGC, R-GCTAAGCAGT TGGTGGTGCA.

cDNA was synthesised as described for RT-qPCR. No pre-amp was performed. Undiluted cDNA, 3 µL, was mixed with 10.0 µL Supermix for Probes (P/N 186-024, Bio-Rad), 1.0 µL TaqMan microRNA Assays (20 ×) and 6.0 µL nuclease-free water. The 96-well plate was sealed and droplets generated by the Automated Droplet Generator and Droplet Generation Oil for Probes (P/N 1864110, Bio-Rad). The cycling condition was 15 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, then 98 °C for 10 min and cooling to 4 °C. Ramp rate was 2 °C/s. A gradient of annealing temperature 56–62 °C was tested. The separation of positive and negative droplets was good at 60 °C, which is the recommended annealing temperature for both Supermix for Probes and the Small RNA Assays (Supplementary Information and Data 1). Droplets were analysed using the QX200 Droplet Reader and the QuantaSoft Analysis Pro software (Bio-Rad). The thresholds for positive droplets were amplitude 3500 for miR371 and 4000 for miR30b across all samples. Triplicate wells were analysed for all samples.
The miR371 and miR30b concentrations from RT-ddPCR are presented as copies/µL serum if not otherwise stated and were calculated from the instrument output given in copies/µL PCR reaction (see Supplementary Information and Data 2).

After injection of 8-cell embryos 2x V2 with antisense oligos (50 pg Lhx1-AS and/or 2.5 ng Fry-MO), embryos were collected at S15 for RNA analysis. The RNA of five embryos was combined and RNA was isolated using the miRVana microRNA Isolation Kit (Ambion). For quantification of mature miRNA expression, miR-199a-3p (hsa-miR-199a), miR-214 (hsa-miR-214), miR-23b (hsa-miR-23b), miR-27b (hsa-miR-27b), miR-24a-3p (hsa-miR-24) and U6 snRNA were reverse transcribed with respective RT primers using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) on a standard thermocycler. Assays for all small RNAs were conducted utilizing respective TaqMan MicroRNA Assays amplified and analyzed on a iQ5 Multi-Color Real-Time PCR with iQ5 Optical System Software version 2.1 (Bio-Rad).