RNA isolation / purification Tissue - Human Brain

Isolating RNA from tissues and paraffin embeded 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 intigrity 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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Discussion

4 years ago

4 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

EZ-RNA Total RNA Isolation Kit

Biological Industries

Upstream tips	Protocol tips	Downstream tips
	- The following might help to increase RNA yield - Heat the DEPC Water to 70°C before adding to the column. - Increase the incubation time to 5 minutes. - Increase the elution volume. - Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration). - Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

Protocol tips
- The following might help to increase RNA yield - Heat the DEPC Water to 70°C before adding to the column. - Increase the incubation time to 5 minutes. - Increase the elution volume. - Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration). - Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

Protocol tips

- The following might help to increase RNA yield

- Heat the DEPC Water to 70°C before adding to the column.

- Increase the incubation time to 5 minutes.

- Increase the elution volume.

- Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration).

- Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

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

NucleoSpin® miRNA

Macherey Nagel

Upstream tips	Protocol tips	Downstream tips
- For quantitative RNA purification from starting material less than 3 mg tissue or 10^6cells, it is advantageous to add 10 μg of Carrier RNA) before binding of small RNA to improve RNA binding. - For animal use mechanical disruption to achieve proper lysis.

Upstream tips
- For quantitative RNA purification from starting material less than 3 mg tissue or 10^6cells, it is advantageous to add 10 μg of Carrier RNA) before binding of small RNA to improve RNA binding. - For animal use mechanical disruption to achieve proper lysis.

Manufacturer protocol Publication protocol 1 Relevant paper

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