RNA isolation / purification Tissue - Human Blood / Serum / Plasma / Buffy coat

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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2 Discussions

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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 4 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

NucleoSpin® RNA Blood

Macherey Nagel

Upstream tips	Protocol tips	Downstream tips
	- To have high yield ,Perform two elution steps with the volume indicated in the individual protocol. About 90–100 % of bound nucleic acids will be eluted - The higher the leukocyte number, the higher the risk to detect residual DNA in the eluted RNA. To avoid this, use less blood.

Protocol tips
- To have high yield ,Perform two elution steps with the volume indicated in the individual protocol. About 90–100 % of bound nucleic acids will be eluted - The higher the leukocyte number, the higher the risk to detect residual DNA in the eluted RNA. To avoid this, use less blood.

Manufacturer protocol Publication protocol 1 Relevant paper

miRNeasy Serum/Plasma Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	- DNase I digestion is not recommended for plasma or serum samples.	- Use 10 mM Tris·Cl,* pH 7.5, not RNase-free water, to dilute the sample before measuring purity

Protocol tips
- DNase I digestion is not recommended for plasma or serum samples.
Downstream tips
- Use 10 mM Tris·Cl,* pH 7.5, not RNase-free water, to dilute the sample before measuring purity

Manufacturer protocol Publication protocol 1 Relevant paper

AllPrep DNA/RNA Micro Kit

Qiagen

Publication protocol 1 Relevant paper

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