RNA isolation / purification Tissue - Human Lymph node

The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.

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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

4 years ago

4 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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RNeasy Plus Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	We employed the same method for RNA isolation for these different tissues as was used for our previously-published brain RNA isolation study, using the RNA EasyPlus Mini Kit (Qiagen, Valencia, CA) (Birdsill et al. 2011). Each tissue sample was dispersed in RNA-lysis buffer (containing 0.1 M β-mercaptoethanol) by mild sonication (10–15 s), and then processed according to the manufacturer’s instructions. In brief, extracted tissue is passed through a prefilter to remove DNA, adjusted with an equal volume of 70 % ethanol and applied to the RNA column. After washes with kit buffers, RNA is eluted from the column with 100 μl of water. A point to note was that for highest yield and integrity, sonication of tissue sample should be carried out for as brief a time as necessary to disrupt the tissue; excessive sonication significantly reduced the yield of RNA from tissues. Some tissues contained insoluble connective tissue or fat that did not dissolve in RNA lysis buffer, but were removed on the DNA prefilters prior to RNA isolation. This same method was used for all cerebellum brain samples.

Protocol tips
We employed the same method for RNA isolation for these different tissues as was used for our previously-published brain RNA isolation study, using the RNA EasyPlus Mini Kit (Qiagen, Valencia, CA) (Birdsill et al. 2011). Each tissue sample was dispersed in RNA-lysis buffer (containing 0.1 M β-mercaptoethanol) by mild sonication (10–15 s), and then processed according to the manufacturer’s instructions. In brief, extracted tissue is passed through a prefilter to remove DNA, adjusted with an equal volume of 70 % ethanol and applied to the RNA column. After washes with kit buffers, RNA is eluted from the column with 100 μl of water. A point to note was that for highest yield and integrity, sonication of tissue sample should be carried out for as brief a time as necessary to disrupt the tissue; excessive sonication significantly reduced the yield of RNA from tissues. Some tissues contained insoluble connective tissue or fat that did not dissolve in RNA lysis buffer, but were removed on the DNA prefilters prior to RNA isolation. This same method was used for all cerebellum brain samples.

Protocol tips

We employed the same method for RNA isolation for these different tissues as was used for our previously-published brain RNA isolation study, using the RNA EasyPlus Mini Kit (Qiagen, Valencia, CA) (Birdsill et al. 2011). Each tissue sample was dispersed in RNA-lysis buffer (containing 0.1 M β-mercaptoethanol) by mild sonication (10–15 s), and then processed according to the manufacturer’s instructions. In brief, extracted tissue is passed through a prefilter to remove DNA, adjusted with an equal volume of 70 % ethanol and applied to the RNA column. After washes with kit buffers, RNA is eluted from the column with 100 μl of water. A point to note was that for highest yield and integrity, sonication of tissue sample should be carried out for as brief a time as necessary to disrupt the tissue; excessive sonication significantly reduced the yield of RNA from tissues. Some tissues contained insoluble connective tissue or fat that did not dissolve in RNA lysis buffer, but were removed on the DNA prefilters prior to RNA isolation. This same method was used for all cerebellum brain samples.

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