Iq5 lightcycler

Total RNA from infected cells was isolated with a Miniprep kit, and small RNA was purified with an RNAiso kit per manufacturer's recommendations (Takara, Dalian, China). Total RNA from archival paraffin sections was isolated using a Mag-Bind^® FFPE RNA Kit (Omega Bio-Tek Norcross, GA, USA). The purified RNA was then used for reverse transcription of the first-strand cDNA synthesis by reverse transcription using M-MLV reverse transcriptase. The expression levels of miRNAs were assessed by the stem-loop RT-PCR method using the Hairpin-it^™ miRNAs. The sequence of the primer used for reverse transcription of mature miR-552 included a stem-loop structure, which was designed based on the sequence of miR-552 (Table 3). The sequence of primer set for amplification of miR-552 and U6 promoter were listed in Table 3. The U6 promoter was included and used to normalize for sample loading and RNA abundance. The qRT-PCR was performed using a Bio-Rad iQ5 lightcycler using a TaKaRa SYBR RT-PCR kit (Takara, Dalian, China). Relative expression was calculated as previously described using real-time PCR efficiencies and the crossing point deviation of unknown sample vs control using the ΔΔ Ct method [43 (link)]. The specificity of the primer sets was determined by sequencing the product of each qRT-PCR reaction.