Taq man microrna reverse transcription kit

HHV-6A or mock-infected BCPAP RNA were obtained by an automated Maxwell RSC-Promega extractor, using the Maxwell RSC miRNA Tissue Kit miRNA (CAT # AS1460, Promega, Sydney, Australia).
Retro-transcription was performed using a Taq-Man MicroRNA Reverse Transcription Kit. TaqMan Individual microRNA assays (Cat. N.4427975, Thermo Fisher Scientific) were used to assess expression of has-miR-9 (assay ID: 000583), hsa-miR-221 (assay ID: 000524), hsa-miR-222 (assay ID: 002276), hsa-miR-146a-5p (assay ID: 000468), hsa-miR-155-5p (assay ID: 002623), and U6 snRNA (assay ID: 001973).
QPCR was performed using an Applied Biosystems Vii A 7 Real-Time PCR (Thermo Fisher Scientific). The relative expression levels of miRNAs were quantified using the 2^∆∆Cq method after normalization for U6 expression, used as housekeeping. All procedures were performed according to the manufacturer’s instructions.

5 µg of total RNA was incubated with or without 3 U/µg RNase R (Epicentre Technologies, Madison, WI, USA) for 15 min at 37 °C. After treatment with RNase R, CircRNA NRIP1 levels were detected by qRT-PCR. Reverse transcriptional reaction was conducted with a PrimeScript™ RT reagent Kit (Promega, Madison, WI) or TaqMan MicroRNA Reverse Transcription Kit (Promega) according to the manufacturer’s manual. qRT-PCR reactions involved employing the Light Cycler-DNA Master SYBR Green qPCR mixture (Bio Rad, Hercules, CA) for miRNA and the Premix Ex Taq™ II (Bio Rad, Hercules, CA) for mRNA with a Biosystems 7500 fast Real-Time PCR System (Thermo, CA, USA). The qPCR amplification conditions included a 5 min denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 60 °C for 1 min. Fluorescent signals were normalized to U6 and GAPDH for miRNA and circRNA, respectively. Then the 2^^−∆∆Ct method was applied to valuate and quantify the relative expression levels. The sequences of primers were shown in the supplementary Table 1.