RNA isolation / purification Tissue - Human Uterus

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

5 years ago

5 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

Aurum™ Total RNA Fatty and Fibrous Tissue Kit

Bio-Rad Laboratories

Upstream tips	Protocol tips	Downstream tips
- Before using lysis solution, add 500 ul of β-mercaptoethanol (β-ME) to the solution for a final concentration of 1%.	- To perform efficient elution preheat the elution solution at 70C in water bath priorto the elution step. - If there is high genomic DNA contamination, increase DNAse I digestion time and use only the DNAse dilution solution provided in the kit	- For better downstream application add appropriate volume of 95-100% ethanol to the wash solutions before intial use. - If elute volume is >80ul, there is a possibility of ethanol contamination. To reduce this, add 1-3min of centrifugation time after the final wash step.

Upstream tips
- Before using lysis solution, add 500 ul of β-mercaptoethanol (β-ME) to the solution for a final concentration of 1%.
Protocol tips
- To perform efficient elution preheat the elution solution at 70C in water bath priorto the elution step. - If there is high genomic DNA contamination, increase DNAse I digestion time and use only the DNAse dilution solution provided in the kit
Downstream tips
- For better downstream application add appropriate volume of 95-100% ethanol to the wash solutions before intial use. - If elute volume is >80ul, there is a possibility of ethanol contamination. To reduce this, add 1-3min of centrifugation time after the final wash step.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy 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

NucleoSpin® RNA

Macherey Nagel

Upstream tips	Protocol tips	Downstream tips
- Aliquot rDNase and store at -20 °C.	- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Upstream tips
- Aliquot rDNase and store at -20 °C.
Protocol tips
- Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.

Publication protocol 1 Relevant paper

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