RNA isolation / purification Tissue - Human Colon

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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 3 matching solutions for this experiment

Upstream tips
- Starting material for RNA purification should be up to 10 mg of a tissue sample.

- Add 10 µl β-ME per 1 ml Buffer TR1 before use.

- To increase RNA yield and to reduce formalin contamination, increase lysate digestion with proteinase K from 15 min at 45°C to 6 hours at 45°C
Protocol tips
- Do not overload the PAXgene RNA MinElute spin column, as this will significantly reduce RNA yield and quality
Downstream tips
- RNA concentration must be measured in 10 mM Tris·Cl, pH 7.5* for accurate quantification
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.
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.
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