RNeasy Micro Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- This kit can be used for the isolation of RNA from up to 5x10^5 cells, or up to 5 mg of tissue.		- Always blank the spectrophotometer with your elution buffer. - When measuring 260 nm absorbance, a unit of 1 on this scale equals to 40 ng of RNA.

Upstream tips
- This kit can be used for the isolation of RNA from up to 5x10^5 cells, or up to 5 mg of tissue.
Downstream tips
- Always blank the spectrophotometer with your elution buffer. - When measuring 260 nm absorbance, a unit of 1 on this scale equals to 40 ng of RNA.

Publication protocol

Total RNA from the various sorted cell populations and bone biopsies was isolated using microfuge columns (MicroColumns, Qiagen). DNase treatment to digest all genomic DNA that could lead to false-positive gene expression results was performed following RNA isolation using Turbo DNA-free DNase (Ambion). RNA quality and purity were confirmed with a Nanodrop spectrophotometer (Thermo Scientific). Since the overall number of the various cell populations was limited (generally <100,000 cells) for the performance of in-depth gene expression analyses, we used the WT-Ovation™ Pico RNA whole transcriptome amplification system (NuGen Technologies, Inc.) to synthesize microgram quantities of amplified cDNA starting with a total RNA input of ~25 ng. In this linear amplification system, the relative representation of each transcript species in the original sample is maintained during and after amplification.

For the QPCR analyses, we designed primers using the Primer Express program (Applied Biosystems). Primer sequences for any of the genes analyzed in this report are available on request. The PCR reactions were run in the ABI Prism 7900HT Real-Time System (Applied Biosystems) using SYBR Green (Qiagen) as the detection method. Normalization for variations in input RNA was performed using a panel of 10 housekeeping genes (18S, G6PDH, GAPDH, GUSB, L13A, RPII, TBP, TUBA1B, Β2M, ACTB) with the geNorm algorithm (http://medgen.ugent.be/~jvdesomp/genorm/) used to select the 3–4 most stable housekeeping genes from the 10 on the plate. The PCR Miner algorithm was used to correct for variations in amplification efficiencies.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Micro Kit below.

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