RNeasy Mini Kit

RNA isolation / purification Cells - immortalized B16

Experiment
RNA isolation / purification Cells - immortalized B16
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
- To yield high 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 extracted using RNeasy Midi Kit (Qiagen, cat. #75144) as described in the following. Mice were euthanized and lungs, liver, and lymph nodes were isolated and snap frozen in liquid nitrogen before being stored at −80°C. For cell cultures, cells were harvested in a good growth phase and 107 cells were pelleted. Organs or cell pellets were mixed with RTL buffer and homogenized using an IKA T10 basic ULTRA-TURRAX homogenizer (Bie & Berntsen). The blender head was washed with 3× PBS, 1× TAE buffer, 1× 70% EtOH, and 1× 96% EtOH between every sample. Samples were then centrifuged for 5 min at 6000 rpm and the supernatant was isolated. For homogenized organs, this step was repeated. Equal volumes of 70% EtOH were added to the supernatant, samples were vortexed, and half of the volume of the samples was added to RNeasy Midi spin columns and centrifuged for 10 min at 5000 rpm. The step was repeated with the last half of the samples. The columns were then washed once in RW1 buffer followed by centrifugation at 5000 rpm for 5 min, and twice in RPE buffer succeeded by centrifugation at 5000 rpm for 2 and 5 min, respectively. The columns were then transferred to new tubes and RNAse free H2O was added to the spin columns. The samples were left to stand for 1 min and then centrifuged at 5000 rpm for 3 min. This step was repeated and total RNA was collected and stored at −80°C. Subsequently, total RNA concentration and purity was measured using a NanoDrop 2000c Spectrophotometer (Thermo Scientific) and the associated computer program NanoDrop 2000.

Full paper   Login or join for free to view the full paper.

Reviews

RNeasy Mini Kit from Qiagen has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

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?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Cells - immortalized B16 using RNeasy Mini Kit from Qiagen.

Paper title
Quantification of B16 Melanoma Cells in Lungs Using Triplex Q-PCR - A New Approach to Evaluate Melanoma Cell Metastasis and Tumor Control
Full paper
Login or join for free to view the full paper.

Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing RNA isolation / purification Cells - immortalized B16 using RNeasy Mini Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms