RNA isolation / purification Tissue - Human Bronchi

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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1 year ago

1 year 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
- Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample.

- Different samples require different methods to achieve complete disruption.

- Incomplete disruption results in significantly reduced nucleic acid yields.

- Overloading the spin columns significantly reduces nucleic acid yields.
Protocol tips
- Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption.

- Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids.

- Excessive homogenization, on the other hand, results in shorter genomic DNA fragments.

- The centrifugation temperature should be 20–25ºC.

-Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.
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.
Upstream tips
- This kit can be used for the isolation of RNA from up to 1x10^6 cells
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