RNeasy Mini Kit

RNA isolation / purification Cells - immortalized A2780

Experiment
RNA isolation / purification Cells - immortalized A2780
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
- To yield hign RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min.

- Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.

- Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

Total RNA was isolated using TRIzol (Gibco, Invitrogen, Grand Island, NY, USA) and purified using RNeasy Mini kit (Qiagen, Hamburg, Germany). The RNA concentration was quantified at 260 nm and 1 μg of RNA was transcribed using SuperScript II (Invitrogen, Carlsbad, CA, USA) reverse transcriptase and oligo(dT) primers (Invitrogen). Quantitative RT-PCRs were performed in duplicates by iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in 30 μl reaction volume containing 1X iQ™ SYBR Green Supermix (0.2 mM dNTP, 3 mM MgCl2), 0.5 μM forward and reverse primer and 2 μl of cDNA. The reaction conditions were: 95°C 3 min, 40 cycles (94°C 30 sec, 55°C 50 sec, 72°C 50 sec), 72°C 7 min followed by melting curve analysis to confirm amplification of the desired single and specific product. The relative expression levels of hypoxia-inducible factor-1α (HIF-1α), Epo, VEGF and β-actin genes (primers designed by SABiosciences) were evaluated using the standard curve method. Standard curves for HIF-1α, Epo, VEGF and β-actin were obtained by amplification of serially-diluted mixtures of cDNA samples (four-fold dilutions), with four to five dilution points, each in duplicate. The calculated resulting relative expression of HIF-1α, Epo, VEGF genes were normalized to relative β-actin expression. The results are presented as mean ± standard deviation of three independent experiments.

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Discussion

Discussion

5 years ago

Author: Switzerland

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

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Papers

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Manufacturer protocol

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

Download PDF Download manufacturer protocol

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