Scribd
Upload
4 views15 pages

Public Lab 7

This document outlines RNA extraction methods, emphasizing the importance of RNA analysis for gene expression and regulation. It details two primary extraction techniques: the TRIzol method, which utilizes a monophasic solution for RNA isolation, and spin column purification, which employs silica columns for selective nucleic acid binding. The document also provides experimental procedures and tips for handling RNA to maintain its integrity during extraction.

Uploaded by

nn6vk49kxp
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
4 views15 pages

Public Lab 7

This document outlines RNA extraction methods, emphasizing the importance of RNA analysis for gene expression and regulation. It details two primary extraction techniques: the TRIzol method, which utilizes a monophasic solution for RNA isolation, and spin column purification, which employs silica columns for selective nucleic acid binding. The document also provides experimental procedures and tips for handling RNA to maintain its integrity during extraction.

Uploaded by

nn6vk49kxp
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 15

Public health and tropical medicine

Lab 7: RNA extraction methods


Fatima Alaa
Lobna Onsi
DNA—--> RNA —---> Protein

● RNA act as the information bridge from DNA to protein.


● RNA analysis is important to get information about gene expression
and regulation.
● RNA isolation is a preliminary step for downstream processes as PCR
and RNA sequencing.
Working with RNA can be tricky because of the following reasons:
1. unstable nature.
2. presence of ribonucleases in cells and the environment can rapidly
degrade RNA.

Tips to be considered during working with RNA

1. Bench and pipettors should be cleaned with RNase decontamination


solution.
2. Start with PPE (personal protection equipment ) by wearing gloves to
protect the samples from RNases found on our skin.
3. Use RNase-free reagents, tubes, and tips
4. Samples should be kept cold throughout the protocol unless it specifically
says to perform a step at room temperature or to apply heat.
There are various approaches used for RNA extraction as:
1. TRIzol
2. Spin column purification
1-TRIzol method
TRIzol Reagent is a monophasic solution of phenol and guanidine isothiocyanate
that facilitate the isolation of RNA.
TRIzol Reagent maintains the integrity of the RNA due to highly effective inhibition
of RNase activity while disrupting cells and dissolving cell components during
sample homogenization.
TRIzol Reagent allows users to perform sequential precipitation of RNA, DNA, and
proteins from a single sample.
After homogenizing the sample with TRIzol Reagent, chloroform is
added, and the homogenate is allowed to separate into :

clear upper aqueous layer Interphase red lower organic layer

(containing RNA) (Containing DNA) (containing the DNA and proteins)


1-Spin column purification method

Column-based extraction is a method that employs selective binding of nucleic acid to a


solid matrix such as silica that is packed in a column.

• The spin columns contain a silica resin that selectively binds DNA and RNA.
• Step 1: A lysis buffer is added to the sample?

To facilitate protein denaturation by altering hydrophobic interactions enabling the


breakdown of cell membranes, hence releasing the target nucleic acid from the cells.

Lysis buffers contain: - chaotropic salts  1) to breakdown Hydrogen bonds and


hydrophobic bonds. 2) to bind the DNA/RNA to silica columns.
- Detergents  to help with protein solublization and lysis
- Enzymes: such as proteinase K  for destruction of proteins in the cell lysate

• Step 2: A binding buffer is added.


By now, the nucleic acid should be bound to the silica columns, while impurities, cellular
proteins and polysaccharides should have passed through.

• Step 3: Adding washing buffers to remove the residual proteins, salts and other
impurities.
- These washing steps are crucial to remove the chaotropic salts to get a high yield and
purity of DNA and RNA.
- Some kits include ethanol in their washing steps to remove all salts bound to the
nucleic acid.
- After washing with ethanol, a centrifugation step is performed to dry the column,
remove the excess ethanol and to a obtain a clean eluate.

- Step 4: Adding an elution buffer to the column: To release the nucleic acid from
the membrane.
To ensure the integrity of the RNA, a sample is load on an agarose gel.
A fragmented RNA will look like this
N.B To ensure having a high yield of nucleic acid, the absorbance of your
sample is measured using a spectrophotometer

Measuring the absorbance requires that you measure your sample at 260 nm and 280 nm
260 nm: is used to measure the amount of RNA in your sample
280 nm: is used to measure the amount of residual proteins and impurities in your sample.

Nucleic acids absorb UV light at 260 nm


Proteins absorb UV light at 280 nm
A ratio of 260/280 is calculated
Acceptable range for a pure RNA is ∼1.8-2.1
If the ratio is less than 1.6 then it indicates the presence of proteins and other contaminants

Conc (ug/ml) =Abs260 xDilution factor x 40


Experimental work
• Each tutorial group will be divided into groups of 3 or 4.
• Some students will handle HCV positive plasma and some will have HCV negative plasma

1- In a clean Eppendorf tube, 400 μl of lysis buffer is added to 400 μl of binding buffer (shake
both before use).

2- Add 200 μl of plasma to the tube.

3- Transfer the contents to a silica spin column and centrifuge at 13-14rpm for 4 minutes.

4- Discard the flow through, add 500 μl of wash buffer 1  centrifuge at 13-14 rpm for 1
minute, discard the flow through  add 500 μl of wash buffer 2 centrifuge at 13-14 rpm for 1
minute, discard the flow through  add 500 μl of wash buffer 3  centrifuge at 13-14 rpm for 1
minute, discard the flow through.
Experimental work cont’d

5- Centrifuge at 13-14rpm for 4 minutes to remove any traces of wash buffer 3.

6- Add 50 μl of Elution buffer, incubate for 4 minutes  centrifuge for 1 minute at 13-14 rpm.

7- Discard silica spin columns.


- 400 ul of lysis buffer To the tube, add Transfer to spin
- 400 ul of binding buffer 200 ul of plasma column

Discard flow through

13-14k
rpm
4 min.

- Discard flow - Discard flow


Discard through through add 500 μl of wash
flow - Add 500 μl of - Add 500 μl of wash buffer 1
13-14k rpm
through 13-14 k rpm
wash buffer 3
13-14 rpm buffer 2 1 min.
1 min. 1 min.

13-14k rpm Blank: 600 ul


4 min. of DPEC water
Discard the Sample: 1 ul of
Incubate for 4 min.
spin columns, RNA stock+
- Add 50 μl of Now you have 599 ul of
Elution buffer your RNA Measure the DEPC water
13-14k rpm
1 min. stock ☺ absorbance

You might also like