RNA isolation / purification Tissue - Rat Brain

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 6 matching solutions for this experiment

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.
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
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.
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.
Magnetic mRNA Isolation Kit

New England BioLabs

Protocol tips
- If the lysate viscous than normal. Pass these cell or tissue lysates several times through a 21-gauge needle. If the yield of mRNA is low Increase the ratio of beads to Lysis/Binding Buffer or add RNase-free Proteinase K to the sample lysate directly before the 10 minute binding incubation
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
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