RNA isolation / purification Tissue - Rat 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

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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 8 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

PAXgene Tissue RNA/miRNA Kit

PreAnalytiX

Upstream tips	Protocol tips	Downstream 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.

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 RNA Isolation Kit

Roche Lifesciences

Upstream tips	Protocol tips	Downstream tips
- To decrease RNase activity,process starting material immeditely or store at 80C. - To increase nucleic yield or purity store all buffer at +15C to +25C.	- To decrease RNase activity,use eluted RNA directly in downstream procedure or store at -80C immediately

Upstream tips
- To decrease RNase activity,process starting material immeditely or store at 80C. - To increase nucleic yield or purity store all buffer at +15C to +25C.
Protocol tips
- To decrease RNase activity,use eluted RNA directly in downstream procedure or store at -80C immediately

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

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

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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