RNA extraction and isolation is a precursor for many methods in molecular biology including Northern Blotting, RT-PCR, and Microarray analysis. This blog post will focus on this precursor method as opposed to other biology techniques on the Scigine blog where I’ve focused on direct analytical techniques. Nonetheless, there is a lot to learn about RNA isolation and plenty of theory that might be applicable in your research.
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While everyone knows DNA is double stranded and helical, few people know that RNA is typically a single stranded polymer. However, secondary structures do emerge within RNA due to complementary base pairing and structures such as tRNA can be double stranded. Also, due to secondary structures, flexible regions of RNA can actually catalyze the cleavage of phosphodiester linkages in nearby RNA chains.
The base polymer looks like this:
The RNA polymer has several ionic groups, inter-chain hydrogen bonds, and it is extremely hydrophilic. All of these forces need to be overcome in order to purify RNA from DNA, carbohydrates, and lipids which have similar structures and physical properties. Polymers of RNA can be short or long, but generally smaller strands that are less than 100 nu cleotide bases are hard to purify because they don’t separate well. Additionally, there are several RNAses present in cells and tissues that can catalyze cleavage of RNA chains, so extreme care needs to be taken to prevent degradation of RNA during RNA isolation and purification procedures.
Typically RNA Extraction procedures start with cell lysis. A buffer that includes Guanidine Thiocyanate or other chaotropes is necessary to mask charges on RNA and water so that the polymer can be purified using solid-phase techniques. Chaotropes linearize the RNA polymer, disrupt hydrogen bonding and destroy the activity of any RNAses present in the cell lysate. Due to their ability to disrupt hydrogen bonding, they also lyse cells by disbanding the phospholipid bilayer.
To facilitate homogenization, a homogenization column (imaged below) may be used. By passing at high speeds through resins, viscous polymers such as DNA and lipids can be separated and the cell lysate will flow more easily.
Ethanol is then added to: reduce the overall water concentration in the sample and precipitate proteins. As you would expect, RNA is highly soluble in water. A spin column (solid-phase support, imaged below) is then used to bind the RNA from the cell lysate.
Initially a low chaotrope concentration wash buffer is used to clean the RNA sample and remove proteins while RNA remains attached to the column.
Next, an ethanol wash removes some of the chaotropic salts that were left over from previous washes.
Finally, without chaotropic salts present, RNAse-free water can be used to elute the RNA sample from the column
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Here’s a technique for RNA Isolation and cDNA preparation from Pancreatic Islets. We can use this technique for analysis of gene expression later on. Our strategy is to use solid-phase extraction of nucleic acids from complex cell lysate samples and then we can prepare the cDNA to examine the expression of genes such as insulin.
Materials for RNA Extraction and cDNA preparation:
Isolated pancreatic islets
RNeasy minikit (#74104 Qiagen)
QIAShredder columns (#79654 Qiagen)
Omniscript reverse transcription kit (#205110 Qiagen)
Oligo dT primers (dilute to 10 µM; #18418012 ThermoFisher Scientific)
Random hexamer primers (dilute to 20 µM; #N8080127 ThermoFisher Scientific)
RNase inhibitor (dilute to 10 units/µl; #N211 Promega)
RNA Extraction/Purification Procedure:
Notes for this RNA Isolation Procedure:
Mr. Young on Google + states:
1. [It is important to] …keep… an amplicon-free, RSase- free, clean environment
2. [Also have] a dedicated space for nucleic acid extraction separate from post amp and Master mix prep
Dr. Carina Jorgensen from Linked In states:
[Check the] integrity of … [your] … RNA … – that is whether or not its degraded – before …[you]…continue with downstream applications…You need to know the quality of RNA to choose your primer for cDNA synthesis, and if you want to continue with real-time qPCR you need to have a certain minimum level when it comes to the quality of your RNA, if you want to be sure that you can trust the following qPCR results. If you want to perform microchip analysis the demand to quality is even higher comparer to qPCR.
HeLA cell RNA Isolation
RNA Extraction from Cumulus Cells
RNA Extraction followed by RT-PCR
RNA Isolation and cDNA Synthesis
Acid-Phenol Extraction of RNA
Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
RNA Extraction Methods in Molecular Biology Book Chapter
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