RNA isolation / purification Tissue - Human Kidney

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.

11 Matching solutions
2 Discussions

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Discussion

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

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 11 matching solutions for this experiment

Absolutely RNA FFPE Kit

Agilent Technologies

Upstream tips	Protocol tips	Downstream tips
	- If RNA degradation is high, ensure that Proteinase K digestion is carried out at 55°C. - If RNA yield is poor ensure that the 90% sulfolane and the pre-filter spin cup filtrate were combined at a 1:1 ratio prior to loading the RNAbinding spin cup.	- If Final RNA concentration is too low for use in subsequent applications use a smaller volume of Elution Buffer, ensuring that the surface of the fiber matrix is completely covered.

Protocol tips
- If RNA degradation is high, ensure that Proteinase K digestion is carried out at 55°C. - If RNA yield is poor ensure that the 90% sulfolane and the pre-filter spin cup filtrate were combined at a 1:1 ratio prior to loading the RNAbinding spin cup.
Downstream tips
- If Final RNA concentration is too low for use in subsequent applications use a smaller volume of Elution Buffer, ensuring that the surface of the fiber matrix is completely covered.

Manufacturer protocol Publication protocol 1 Relevant paper

PureLink™ FFPE RNA Isolation Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	- To increase RNA concentration, reduce elution volume. - Performing a second elution step increases the RNA yield by up to 30%. - Pre-warming the elution buffer to 65°C increases the RNA yield by up to 15-20%	- The purified RNA is suitable for downstream applications such as reverse transcription, RT-PCR, and real-time quantitative RT-PCR (qRT-PCR).

Protocol tips
- To increase RNA concentration, reduce elution volume. - Performing a second elution step increases the RNA yield by up to 30%. - Pre-warming the elution buffer to 65°C increases the RNA yield by up to 15-20%
Downstream tips
- The purified RNA is suitable for downstream applications such as reverse transcription, RT-PCR, and real-time quantitative RT-PCR (qRT-PCR).

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

RNeasy MinElute Cleanup Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
- This kit purifies RNA from up to 45 mg tissue.	- Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C.	- If there is low A260/A280 value use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before measuring purity.

Upstream tips
- This kit purifies RNA from up to 45 mg tissue.
Protocol tips
- Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C.
Downstream tips
- If there is low A260/A280 value 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

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

miRNeasy 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

High Pure FFPET RNA Isolation Kit

Roche Lifesciences

Upstream tips	Protocol tips	Downstream tips
	- For better RNA yield, Store all buffers at +15 to +25°C	- To promote selective binding of RNA to the glass fibers add 0.5 volume of absolute ethanol to the lysate.

Protocol tips
- For better RNA yield, Store all buffers at +15 to +25°C
Downstream tips
- To promote selective binding of RNA to the glass fibers add 0.5 volume of absolute ethanol to the lysate.

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

Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
- This kit purifies RNA from up to 35 mg tissue	- Do not extend the 15 min incubation at 80°C more than 2 min

Upstream tips
- This kit purifies RNA from up to 35 mg tissue
Protocol tips
- Do not extend the 15 min incubation at 80°C more than 2 min

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

ExpressArt FFPE Clear RNAready

Amsbio

Upstream tips	Protocol tips	Downstream tips
- This kit purifies RNA from up to 30 mg tissue

Upstream tips
- This kit purifies RNA from up to 30 mg tissue

Manufacturer protocol Publication protocol 1 Relevant paper

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