PicoPure™ RNA Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Can be used when there is a very less amount of starting material.

Upstream tips
- Can be used when there is a very less amount of starting material.

Publication protocol

RNA was isolated with the TRIzol® reagent (Invitrogen) or with the ARCTURUS® PicoPure® RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.

Isolated (and immune cell depleted) native glioma
cells (7.0´104
up to 2.5´105
) were seeded in a six-well
plate (Thermo Fisher Scientific) onto a cell culture insert
with a membranous bottom (pore size: 8 µm; Merck
Millipore). While the growth medium [Dulbecco’s
modified Eagle’s medium (DMEM); Invitrogen] in the
upper chamber above the membrane contained only 2% of fetal bovine serum (FBS; Invitrogen), the medium below the membrane contained 10% FBS, favoring migration of fast-migrating cells into the lower cell chamber. The migration process was stopped after 24 h, and migrated cells were collected from the lower chamber and the bottom side of the insert by a cell scraper (fast-migrating fraction). In addition, nonmigrated cells were collected from the upper chamber as well (slowmigrating fraction). Cells were harvested by centrifugation at 1,400 rpm for 5 min. RNA was isolated using the ARCTURUS® PicoPure® RNA Isolation Kit (Thermo Fisher Scientific).

Glioma stem-like cells were generated from the human glioblastoma cell lines (A172, T98G, and U251MG) by sequential cultivation in neurosphere medium17 plus 20 ng/ml basic fibroblast growth factor (bFGF; ImmunoTools, Friesoythe, Germany) and 20 ng/ml epidermal growth factor (EGF; PeproTech, Rocky Hill, NJ, USA)16,18–20. Developing glioma spheroids were kept for 6 weeks with a dissociation procedure by trypsinization every other week. To verify whether stem-like cells have been successfully generated, one fraction was differentiated in stem cell medium containing 10% FBS without additional growth factors for 9 days, RNA was isolated
using the ARCTURUS® PicoPure® RNA Isolation Kit,
and transcription of SOX2, PROM1, MSI1, NES, and
CXCR4 was determined in both glioma stem-like spheroids and differentiated fractions by qRT-PCR.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for PicoPure™ RNA Isolation Kit below.

Download manufacturer protocol

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