RNA isolation / purification Tissue - Human Joints

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

3 Matching solutions
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Discussion

4 years ago

4 years 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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Discussion

5 years ago

5 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

RNeasy Fibrous Tissue Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
- This kit purifies RNA from up to 30 mg tissue		- If there is low RNA yield,Repeat RNA elution, incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - If there is low Low A260/A280 value use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity. - If RNA does not perform well in downstream experiments this could be due to salt carryover dyuring elution step, so ensure that Buffer RPE is at 15–25°C.

Upstream tips
- This kit purifies RNA from up to 30 mg tissue
Downstream tips
- If there is low RNA yield,Repeat RNA elution, incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. - If there is low Low A260/A280 value use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity. - If RNA does not perform well in downstream experiments this could be due to salt carryover dyuring elution step, so ensure that Buffer RPE is at 15–25°C.

Manufacturer protocol Publication protocol 1 Relevant paper

TRI Reagent® Sigma

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Upstream tips
- 1-Bromo-3-chloropropane is less toxic than chloroform and its use for phase separation decreases the possibility of contaminating RNA with DNA - The chloroform used for phase separation should not contain isoamyl alcohol or other additives.

Manufacturer protocol Publication protocol 1 Relevant paper

TRIzol Reagent

Thermo Fisher Scientific

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

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

Manufacturer protocol Publication protocol 1 Relevant paper

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