RNA isolation / purification Tissue - Rat Spinal cord

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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1 year ago

1 year 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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2 years ago

2 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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TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
The dorsal horn of the spinal cord at the sacral and coccyx levels was homogenized in 1 ml of TRIzol reagent per 50 to 100 mg of tissue sample using TissueLyser (Qiagen). The samples were shaken at high speed in 2-ml round-bottomed microcentrifuge tubes with stainless steel beads for 15 min at room temperature. Following homogenization, the samples were centrifuged at 1200g for 5 min at 4°C, and the fatty layer was discarded. The cleared supernatant was transferred to a new microcentrifuge tube (Qiagen). For phase preparation, 200 μl of chloroform was added per 1 ml of TRIzol reagent, mixed for 15 s, and incubated for 2 min at room temperature. The mixture was then centrifuged at 13,300 rpm for 15 min at 4°C. The colorless upper aqueous phase containing RNA was transferred to a fresh tube and mixed with 600 μl of 70% molecular grade ethanol.
Upstream tips
C6, PC12 and CGC cultures were washed with 1× Phosphate-Buffered Saline (PBS) prior to RNA isolation.
Protocol tips
The cells were lysed directly on the culture dish using the lysis buffer provided in Qiagen’s RNeasy Mini Plus kit for total RNA isolation, and genomic DNA was removed using Qiagen’s gDNA Eliminator columns following the manufacturer’s protocols (Qiagen, Toronto, ON, Canada). Total RNA from juvenile rat hippocampus was extracted using TRIzol (Invitrogen, Burlington, ON, Canada), and further purified using Qiagen’s RNeasy Mini Plus kit and gDNA Eliminator columns following the manufacturer’s protocols.
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