RNA isolation / purification Tissue - Rat Heart

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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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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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 6 matching solutions for this experiment

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- 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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

MagMAX™-96 Total RNA Isolation Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
- This kit can be used for the isolation of RNA from ≤ 5 mg animal tissue.	- To avoid DNA contamination, either reduce sample size, or include an additional DNase treatment step after RNA isolation	- If there is low yield and/or inconsistent yield, lowering the amount of sample input may improve results

Upstream tips
- This kit can be used for the isolation of RNA from ≤ 5 mg animal tissue.
Protocol tips
- To avoid DNA contamination, either reduce sample size, or include an additional DNase treatment step after RNA isolation
Downstream tips
- If there is low yield and/or inconsistent yield, lowering the amount of sample input may improve results

Manufacturer protocol Publication protocol 1 Relevant paper

PAXgene Tissue RNA/miRNA Kit

PreAnalytiX

Upstream tips	Protocol tips	Downstream tips
	- Add β-Mercaptoethanol (β-ME) to Buffer TM1 before use. - Buffer TM1 and Buffer TM2 may form a precipitate during storage. If necessary, warm to 37°C to dissolve - Before using carrier RNA for the first time, dissolve it (310 µg) in 1 ml RNase-free water.

Protocol tips
- Add β-Mercaptoethanol (β-ME) to Buffer TM1 before use. - Buffer TM1 and Buffer TM2 may form a precipitate during storage. If necessary, warm to 37°C to dissolve - Before using carrier RNA for the first time, dissolve it (310 µg) in 1 ml RNase-free water.

Manufacturer protocol Publication protocol 1 Relevant paper

miRNeasy FFPE Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
- To allow complete penetration by formalin, use tissue samples less than 5 mm thick	- Perform all centrifugation steps using a microcentrifuge placed at 15–25°C.	- Do not exceed fixation time of 24 hours as it results in poor performance in downstream assay.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

TRI Reagent® MRC

Molecular Research Center, Inc.

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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