ISOLATION OF TOTAL RNA

Isolation of total RNA is typically a preliminary step in many research settings.

Find out and provide me three different purposes for isolating RNA
from cells. One reason may be found in the video you may watch from
the link below.
Total RNA extraction - YouTube
More care must be taken in isolating RNA then DNA. Explain
3 reasons for the easier degradation of RNA compared to
DNA.

Suggest 3 precautions to protecting against RNA degradation.

 3. Summarize the steps and the functions of the chemicals used in RNA extraction.
You may use flow charts, diagrams and/or tables.

Note you are using filter Based RNA

Isolation

Benefits
Convenience and ease of use
Ability to isolate RNA and DNA
Ability to manufacture membranes of
various dimensions

Drawback
Propensity to cog with particulate material
Retention of large nuclei acids such as
gDNA
Checking RNA quantity and purity
The traditional and least expensive way to determine RNA quantity and
purity is to measure UV absorption of the sample using a
spectrophotometer.
A260 for quantification: RNA has a maximum absorption at 260nm and RNA
concentration is determined by the OD reading at 260nm as given by the
following conversion: an A260 of 1.0 is equivalent to 40 µg/mL of RNA.
A280 for protein contamination:
In addition to the OD260, measurements should also be taken at 280nm and
230nm.  The A260/A280 ratio is an indication of the level of protein
contamination in the sample.  Pure RNA has an A260/A280 ratio of 2.1,
however values between 1.8-2.0 are considered acceptable for many
protocols.
A230 for other stuff: In addition to protein contaminants, RNA preparations
can also contain contaminants such as guanidine salts and phenol (used in
RNA isolation protocols).  A high peak at A230 indicates contamination with
either of these. The ideal A260/A230 ratio is greater than 1.5.
 Determine the RNA concentration

Note: Be aware that any contaminating DNA in the RNA prep will lead to an
overestimation of yield, since all nucleic acids absorb at 260 nm.

Even if an RNA prep has a ratio outside of this range, it may function well in
common applications such as RT-PCR, Northern blotting, and RNase protection
assays.
How to use a spectrophotometer?

Watch the youtube https://www.youtube.com/watch?v=EvDwmgSbnnk, and

state the main steps.

Use the Simulator of ultraviolet absorption spectra for proteins and nucleic acids at
http://biomodel.uah.es/en/lab/abs/espectro.htm

Draw a table of the % Protein, % DNA and A260/A280 values for pure DNA, pure protein
and the 4 samples.
RNA extraction using
Kit A and Kit B were
compared. Compare
the parameters
measured to
recommend which Kit
to use
Checking RNA Integrity

Degraded RNA doesn’t perform well in downstream applications,

therefore it is important to check the integrity of your RNA preparation.

The least expensive method for checking RNA integrity is to run the RNA
on a 1% standard agarose gel and examine the ribosomal RNA (rRNA)
bands (formaldehyde gels are not required for this quick assessment).

Agarose gel sieves

DNA fragments
according to size
– Small fragments
move farther
than large
fragments Gel running

Use a 3% gel to
separate small
fragment sizes.
Lower concentration
gels can separate
Before being loaded on to a gel, the RNA is mixed with a sample buffer. This buffer
consists of a viscous liquid such as glycerol, sucrose or Ficoll, and the dye
bromophenol blue. The sample buffer may contain another dye, xylene cyanol FF,
in addition to the bromophenol blue. The purpose of this buffer is to make
the RNA sample dense so that it sinks to the bottom of the well. The dyes, in
addition to colouring the sample to facilitate loading, are used to monitor the
progress of the electrophoresis.
This simulation introduces gel electrophoresis, a technique used to separate biological
molecules. In this simulation, gel electrophoresis is used to separate dyes and visualize
them in an agarose gel.

https://www.labxchange.org/library/items/lb:LabXchange:9548bee3:lx_simulation:1

Take a screen shot of the gel in the correct

orientation
Take a screen
shot to show
the tubes in a
balanced
position.
Use the dropdown menu to attach
the leads to the power supply and
gel electrophoresis box.
Complete the electrophoresis, and take a screenshot of the gel
for your journal.

Do bigger or smaller nucleic acid molecules be seen nearer to the

positive electrode?
All the RNA from a cell is referred to as "total RNA". Total RNA mainly contains
three types of specialized RNA molecules. They are messenger RNA (mRNA),
transfer RNA (tRNA), and ribosomal RNA (rRNA). Each of these RNA types has a
unique structure related to its function.

The vast majority (~85%) of RNA in the cell is rRNA, and this is what can be seen
on a gel after size separation by denaturing gel electrophoresis. The main rRNA
components of the two ribosomal subunits form the bands that are usually
predominant in the gel. mRNA is present, but it appears as a hazy background
smear because of its variable size (0.5-10 kb) and its relatively small
representation (~2%) in total RNA. tRNA and other small RNA molecules makes
up 10-15% of total RNA but are not efficiently resolved on denaturing agarose
gels.
Intact total RNA run on a denaturing gel will have sharp, clear rRNA bands (e.g.,
28S and 18S in mouse and rat). Although they are present in equimolar amounts,
the 28S rRNA band is twice the size of the 18S rRNA band. Thus it will be
approximately twice as intense as the 18S rRNA band when stained with ethidium
bromide. This 2:1 ratio (28S:18S) is a good indication that the RNA sample is
completely intact.

Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA
bands, or will not exhibit the 2:1 ratio of high quality RNA.

Completely degraded RNA will appear as a very low molecular weight smear.
Inclusion of RNA size markers on the gel makes it possible to determine the
size of any bands or smears to be determined and will also serve as a good
control to ensure the gel was run properly. In this experiment, RNA marker
(from Promega) will be run together with total RNA samples in the non-
denaturing agarose gel that does not contain any denaturant such as
formaldehyde.
 The upper ribosomal band (28S in eukaryotic
cells and 23S in bacterial cells) should be about
twice the intensity of the lower band (18S in
eukaryotic cells and 16S in bacterial cells) and
should be crisp and tight.  If the rRNA bands are
of equal intensity, then it suggests that some
degradation has occurred.  mRNA runs
between the 2 ribosomal bands and might be
seen as a smear.  This is ok. However, smearing
below the rRNA bands suggests that you have
poor quality RNA.  You also should not see
higher molecular weight bands that might
indicate that the RNA is contaminated with
DNA.
Should the above gel be run together with a RNA marker lane? Why?
Summarizing main points.

After you have completed RNA (or DNA) extraction,

how would you determine if you were successful and
could continue with your experiment for example
sequencing?