NucleoSpin® miRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- For quantitative RNA purification from starting material less than 3 mg tissue or 10^6cells, it is advantageous to add 10 μg of Carrier RNA) before binding of small RNA to improve RNA binding. - For animal use mechanical disruption to achieve proper lysis.

Upstream tips
- For quantitative RNA purification from starting material less than 3 mg tissue or 10^6cells, it is advantageous to add 10 μg of Carrier RNA) before binding of small RNA to improve RNA binding. - For animal use mechanical disruption to achieve proper lysis.

Publication protocol

Total RNA was isolated from cultured cells or tumor tissues by using a NucleoSpin microRNA isolation kit (TaKaRa, Otsu, Japan). RNA concentrations and purity were assessed by UV spectrophotometry. RNA integrity was checked by formaldehyde gel electrophoresis. To determine the expression levels of miR-124 and miR-133b, we conducted quantitative RT-PCR (qRT-PCR) by using TaqMan MicroRNA Assays (Applied Biosystems) and THUNDERBIRD Probe qPCR Mix (TOYOBO Co.,LTD.,Osaka Japan) according to the manufacturer's protocol. RNU6B was used as an internal control. For determination of the expression levels of PTB1, PKM1, PKM2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs, total RNA was reverse-transcribed with a PrimeScript® RT reagent Kit (TaKaRa). Real-time PCR was then performed with primers specific for them by using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The primers for PTB1, PKM1, PKM2, and GAPDH were the following: PTB1-sense, 5′-ATC AGG CCT TCA TCG AGA TGC ACA-3′, and PTB1-antisense, 5′-TGT CTT GAG CTC CTT GTG GTT GGA-3′; PKM1-sense, 5′-CGA GCC TCA AGT CAC TCC AC-3′, and PKM1-antisense, 5′-GTG AGC AGA CCT GCC AGA CT-3′24; PKM2-sense, 5′-ATT ATT TGA GGA ACT CCG CCG CCT-3′, and PKM2-antisense, 5′-ATT CCG GGT CAC AGC AAT GAT GG-3′24; GAPDH-sense, 5′-CTC AGA CGG CAG GTC AGG TCC ACC-3′, and GAPDH-antisense, 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′. GAPDH was used as an internal control. The primers for both PKM1 and PKM2 were used after confirmation that efficient CT values had been obtained from standard curves. The relative expression levels were calculated by the ΔΔCt method.

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Manufacturer protocol

Download the product protocol from Macherey Nagel for NucleoSpin® miRNA below.

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