Total RNA Extraction From Tissue
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Objectives
Isolation of total RNA from blood sample.
RNA Isolation
RNA Isolation
RNA (Ribonucleic acid).
RNA, is
formed from DNA by a process called transcription.
The three most well-known types of RNA: mRNA, rRNA, and tRNA.
Obtaining pure RNA is an essential step ?
Types of RNA isolation?
What are Ribosomes? | Ribosome Function and Structure
https://www.youtube.com/watch?v=WjnUJusSYAo
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https://www.biochain.com/blog/types-of-rna-and-how-to-extract-or-purchase-it/
https://www.biochain.com/blog/types-of-rna-and-how-to-extract-or-purchase-it/
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The basic procedures used in RNA purification are similar to those of DNA,
Why?
However, RNA isolation is more difficult than DNA purification,
Why?
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Organic extraction methods
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Filter- based spin basket formats
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Magnetic particle methods.
The GOAL is to..
extract pure intact RNA
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lower organic phase
middle phase that contains denatured proteins and gDNA
upper aqueous phase contains RNA
https://www.youtube.com/watch?v=Vz2uu5iyT0I
https://www.youtube.com/watch?v=Vz2uu5iyT0I
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https://www.youtube.com/watch?v=SJ6c40lJuK0
https://mvspacific.com/
REAGENTS/DNA%20Isol
ation%20Kits.htm
Practical Part
Aim:
•
To isolate pure and intact total RNA from Rat blood sample using Filter- based
spin basket format.
Principle
:
:
•
In general, Lysing and denaturing cells to liberate total nucleic acids in the presence of
RNase inhibitory agents
. Additional steps are then required to remove DNA.
Principle of
Principle of
:
:
F
F
ilter-based, spin basket method
11
Principle
of Gene JET RNA Purification Kit:
Samples are lysed in Lysis Buffer, which contains guanidine thiocyanate, a chaotropic salt capable of protecting RNA
from endogenous RNases.
The lysate is then mixed with ethanol and loaded on a purification column.
The chaotropic salt and ethanol cause RNA to bind to the silica membrane while the lysate is spun through the
column.
Subsequently, impurities are effectively removed from the membrane by washing the column with wash buffers.
Pure RNA is then eluted under low ionic strength conditions with nuclease-free water.
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The
concentration, purity, and integrity
of the extracted RNA need to be determined.
•
The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8–2.1 indicates
highly purified RNA.
This content covers the isolation of total RNA from tissue samples, focusing on the methods, principles, and objectives involved in RNA extraction. It discusses the challenges of RNA purification compared to DNA, different types of RNA, including mRNA, rRNA, and tRNA, as well as the principles behind organic extraction and magnetic particle methods for RNA isolation. Additionally, the practical part details the aim and principle of isolating pure and intact total RNA from a rat blood sample using a filter-based spin basket format.
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Presentation Transcript
Objectives Isolation of total RNA from blood sample.
RNA Isolation RNA (Ribonucleic acid). RNA, is formed from DNA by a process called transcription. The three most well-known types of RNA: mRNA, rRNA, and tRNA. Obtaining pure RNA is an essential step ? Types of RNA isolation?
mRNA, tRNA, and rRNA function | Types of RNA : Types of RNA https://www.youtube.com/watch?v=1THyMOk3WU0 What are Ribosomes? | Ribosome Function and Structure https://www.youtube.com/watch?v=WjnUJusSYAo https://www.biochain.com/blog/types-of-rna-and-how-to-extract-or-purchase-it/
Method of RNA Isolation The basic procedures used in RNA purification are similar to those of DNA, Why? However, RNA isolation is more difficult than DNA purification, Why? Removal of proteins and contaminants Recovery of RNA Lysis
Method of RNA Isolation Organic extraction methods The GOAL is to.. Filter- based spin basket formats extract pure intact RNA Magnetic particle methods.
Principles of Organic extraction method Solubility dependence lower organic phase middle phase that contains denatured proteins and gDNA upper aqueous phase contains RNA https://www.youtube.com/watch?v=Vz2uu5iyT0I https://www.youtube.com/watch?v=Vz2uu5iyT0I
Principles of Magnetic particle method https://mvspacific.com/ REAGENTS/DNA%20Isol ation%20Kits.htm https://www.youtube.com/watch?v=SJ6c40lJuK0
Aim: To isolate pure and intact total RNA from Rat blood sample using Filter- based spin basket format. Principle: In general, Lysing and denaturing cells to liberate total nucleic acids in the presence of RNase inhibitory agents . Additional steps are then required to remove DNA.
Principle of: Filter-based, spin basket method 11
Principle of Gene JET RNA Purification Kit: Samples are lysed in Lysis Buffer, which contains guanidine thiocyanate, a chaotropic salt capable of protecting RNA from endogenous RNases. The lysate is then mixed with ethanol and loaded on a purification column. The chaotropic salt and ethanol cause RNA to bind to the silica membrane while the lysate is spun through the column. Subsequently, impurities are effectively removed from the membrane by washing the column with wash buffers. Pure RNA is then eluted under low ionic strength conditions with nuclease-free water.
Results: The concentration, purity, and integrity of the extracted RNA need to be determined. The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8 2.1 indicates highly purified RNA.
