RNA isolation / purification Tissue - Mouse 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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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 10 matching solutions for this experiment

mirVana™ miRNA Isolation Kit, with phenol

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation - To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step	- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA

Protocol tips
- For RNA to dissolve better and to yeild high levels, preheat elution buffer at 95C. - Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation - To capture more amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step
Downstream tips
- Do vortex for 2-plus minutes during Acid-Phenol:Chloroform extraction step. This will facilitate the removal of tightly bound proteins typically associated with RNA

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Plus Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- On-column digestion is preferable - For high gDNA elimination spin at 6000rpm for 5 min instead of the quick high speed spin	- 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
- On-column digestion is preferable - For high gDNA elimination spin at 6000rpm for 5 min instead of the quick high speed spin
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

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

Total RNA Purification Kit

Norgen Biotek

Upstream tips	Protocol tips	Downstream tips
- For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed		- Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.

Upstream tips
- For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed
Downstream tips
- Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.

Manufacturer protocol Publication protocol 1 Relevant paper

TriPure Isolation Reagent

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
- Process tissues immediately and freeze at -60c or below for Low RNA degradation	- Adding too little reagent can lead to contamination with DNA

Upstream tips
- Process tissues immediately and freeze at -60c or below for Low RNA degradation
Protocol tips
- Adding too little reagent can lead to contamination with DNA

Manufacturer protocol Publication protocol 1 Relevant paper

miRNeasy Mini kit

Qiagen

Upstream tips	Protocol tips	Downstream 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.

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.

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

Isolate II RNA Mini Kit

Bioline

Upstream tips	Protocol tips	Downstream tips
- This kit can be used for the isolation of RNA from up to 1x10^6 cells

Upstream tips
- This kit can be used for the isolation of RNA from up to 1x10^6 cells

Manufacturer protocol Publication protocol 1 Relevant paper

SV Total RNA Isolation System

Promega

Upstream tips	Protocol tips	Downstream tips
	-Do not freeze-thaw the DNase more than three times after rehydration

Protocol tips
-Do not freeze-thaw the DNase more than three times after rehydration

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Lipid Tissue Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Manufacturer protocol Publication protocol 1 Relevant paper

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