RNA isolation / purification Tissue - Human Liver

Isolating RNA from tissues and paraffin-embedded tissue samples can be challenging due to cross-linking of biomolecules and fragmented nucleic acids. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in presence of RNAse inhibitors. The homogenization process should be carried out on dry ice to maintain the integrity of RNA.

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1 year ago

1 year ago by Paul G. Macon United States

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

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2 years ago

2 years ago by Aaron Stege Netherlands

Problem in phase separation after using serum/plasma kit

I used a serum/plasma kit for my serum samples. After the phase separation the samples should have 3 phases: a colourless aqueous phase, a white interphase and a red organic phase. However, in some of my samples there was no aqueous phase unless I wait for an extended period of time. How can I circumvent this problem?

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Found 7 matching solutions for this experiment

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.
Upstream tips
- When processing <2 µg tissue, carrier RNA may be added to the lysate. Foaming can be reduced by adding Reagent DX at a final concentration of 0.5% (v/v) before disruption and homogenization.

- Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution.
before homogenization
Upstream tips
- To allow complete penetration by formalin, use tissue samples less than 5 mm thick
Protocol tips
- Perform all centrifugation steps using a microcentrifuge placed at 15–25°C
Downstream tips
- Do not exceed fixation time of 24 hours as it results in poor performance in downstream assay
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
NucleoSpin® RNA

Macherey Nagel

Upstream tips
- Aliquot rDNase and store at -20 °C.
Protocol tips
-Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.
Protocol tips
- To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
Upstream tips
- Do not freeze-thaw the DNase more than three times after rehydration
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