RNA isolation / purification Cells - immortalized SH-SY5Y

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

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1 year ago

1 year ago by Ralf Friedmann 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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Found 4 matching solutions for this experiment

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C.
- Addition of β-mercapto ethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation.

- To capture more amount of mRNA, modify the amount of lysis buffer:ethanol (from 1:1 to 1:1.5) during the binding step.
Downstream tips
- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA.
Protocol tips
Total RNA from the cells was isolated using a FujiFilm
QuickGene 810 machine with QuickGene RNA cultured
cell HC kit S (FujiFilm LifeScience, FujiFilm, Tokio,
Japan). The amount and quality of isolated RNA were
measured on NanoDrop (A260/A280 and A260/A230)
(Thermo Scientific, Massachusetts, USA) and the
quality (RIN number) was checked using Agilent RNA
chips (Agilent, Colorado, USA).
PureLink™ RNA Mini Kit

Thermo Fisher Scientific

Protocol tips
- Depending on the material you want to isolate RNA from, please refer to the manual for additional reagents/lab equipment that are required
Downstream tips
- DNase is not supplied with this kit, a DNase kit is available which is compatible with this kit
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
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