RNA isolation / purification Tissue - Human Lung

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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Protocol tips
Whole lung tissue was homogenized prior to RNA extraction (BeadBlaster 24, Benchmark Scientific) in the presence of lysis buffer (RLT Plus, Qiagen). Total RNA from whole lung tissue and sorted alveolar macrophages and alveolar type II cells was extracted (AllPrep DNA/RNA Mini Kit, Qiagen) and quality and quantity were assessed (TapeStation 4200, Agilent).
Protocol tips
For the cell lines, RNA was extracted from the cells using the RNEasy® kit (Qiagen), generating high‐quality total RNA (i.e., RIN > 8) that was used as input material for library construction with Illumina TruSeq Stranded mRNA reagents.
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