RNeasy Protect Animal Blood Kit (50)

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Publication protocol

RNA extraction and quantitative real-time PCR (qPCR):Total RNA was isolated from cultured cells by treatment with the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and from blood specimens of wild-type (n = 9) and mutant-type (n = 9) piglets by use of the RNeasy Protect Animal Blood Kit (Qiagen). The isolated RNA samples were reverse-transcribed into cDNA with the miScript II RT Kit (Qiagen). The samples were subjected to qPCR in the 7900HT Standard Real-Time PCR System (Applied Biosystems Inc., Hercules, CA, USA) and using gene-specific primers and reagents from the miScript SYBR Green PCR Kit (Qiagen). Mature miR-15b-5p, miR-15b-3p and miR-16 were detected by the forward primers miR-15b-5pF, miR-15b-3pF and miR-16F, respectively (S1 Table), with the MiScript Universal Primer (of the miScript II RT Kit) used as the reverse primer for all. Pri-miR-15b-F1 and pre-miR-15b-R (S1 Table) were used for detection of pri-miR-15b. Pre-miR-15b-F2 (S1 Table) and precursor-miR-15b-R were used to detect the total expression level of pri-miR-15b and pre-miR-15b. The quantified expression levels of the various miRNAs and the miR-15b precursors were normalized according to the level of mCherry gene detected by qPCR as red fluorescent protein expressed from the pmR-mCherry vector. Relative abundance of detected transcripts was calculated using the 2−ΔΔCt method.

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