Objectives:-

1. To determine the effect of phenol-chloroform based method in RNA isolation.

2. To determine integrity purity and concentration of RNA.

Introduction:

Complete set of RNA in a cell is called total RNA. This set involves all RNA types, including
mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA) and other minor RNA species, like small RNA
and lncRNA. Although whole blood is a readily available source for biological sampling, RNA
extraction from blood is often difficult. White blood cell which is the most valuable RNA containing
cells are outnumbered by red cells approximately 1000:1. In addition, once blood is collected,
expression levels are exposed by a variety of ex vivo changes, including degradation by potent
RNases. Isolation of RNA requires cautious handling of samples and good aseptic techniques. It
is important to use only RNase-free solutions during the extraction, as well as RNase-free pipet
tips and glassware. Several methods are used in molecular biology to isolate RNA from samples,
the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter
paper based lysis and elution method features high throughput capacity. RNA extraction in liquid
nitrogen, commonly using a mortar and pestle or specialized steel devices known as tissue
pulverizers is also useful in preventing ribonuclease activity.

Challenges of isolating, storing and analyzing RNA

Blood is one of the most commonly used sample types for RNA isolation because of its
accessibility and regenerative properties. It poses unique challenges for the isolation of high
quality RNA although blood is easier to process than solid tissues because it does not require
mechanical disruption. There are some challenges when isolating RNA from human blood sample.
Firstly, blood has a complex cellular composition. The complex cellular nature of blood makes it
difficult to detect differential gene expression that occurs in only a subset of cell types. The RNA
containing cells of interest, leukocytes, comprise <1% of the cell mass. More than 99% of the
cellular blood fraction is made up of red blood cells, including immature reticulocytes, which
contain high levels of globin mRNA. Globin mRNA can compromise the detection of other specific
mRNAs from leukocyte.

Blood contains biochemical that can compromise downstream applications. Enzyme-

inhibiting compounds, such as RNase, anticoagulants, and heme, are present at relatively high
concentrations in blood compared to many other tissues. They can significantly impair
downstream analyses such as microarray analysis and RT-PCR if they are not sufficiently removed
during RNA isolation. Followed by, blood is often collected at clinical sites but processed for RNA
isolation elsewhere. This leads to the requirement for RNA stabilization prior to shipping and RNA
purification. For short term storage, the RNA sample can be stored at -20° C generally carried
out after the snap freeze. For long-term storage, the RNA sample can be stored at -80° C. The
RNA samples should be stored in aliquots in several tubes. This would reduce the repeated
thawing of freezes RNA for different experiments. Thawing should be done with great care. The
sample should not place at room temperature immediately. The temperature should be increased
slowly to prevent the damage to the RNA. RNA Stabilization solutions can be used which provides
more flexibility and time to allow the researcher to postpone RNA isolation for days, weeks, or
even months after tissue collection, without sacrificing the integrity of the RNA.

There are some precautions need to be concerned during RNA isolation. The isolation of
the RNA or DNA should be done in the aseptic condition and with a great care. When handling
the reagents and RNA samples, it is important to wear gloves. Besides this, gloves should change
frequently with 70% ethanol. Use of the solution that can completely remove RNase
contamination from the equipment and workspace. RNase Zap is the solution commonly used to
prevent RNase contamination. Alkaline pH, high temperatures, and heavy metal ions should be
avoided when possible. Hands may contain nuclease enzymes which can damage the RNA and
also DNA. So, it is important not to touch Eppendorf tube and micropipette’s tip.

Materials and Methods

RNA isolation from human blood using phenol-based method:

1. Blood in Heparin containing vacutainer is collected

2. Whole blood 1:1 using 2X Extraction Buffer (final buffer concentration is 10 mM

Tris, 1 mM EDTA and 1% SDS) is diluted.

3. Inverting the tube several times to mix the homogeneity

4. Proteinase K to a final concentration of 300 μg/ml is added and mixed well. Followed
by, incubate 3 to 6 hour at 37°C. Then solution is cooled to room temperature
 5. Phenol-TE extraction is repeated to the aqueous phase.

6. An equal volume of Phenol-TE is added, well mixed by inversion centrifuge at 4,300 X g

for 15 min to separate the phases and save the upper (aqueous) phase.

7. 1 volume of SEVAG is added, well mixed to the aqueous phase centrifuged at 4,300 X g
for 15 min to separate the phases and save the upper (aqueous) phase.

8. 0.1 volume 2 M NaCl (final concentration is approximately 0.2 M) is added and mixed
well by inversion to the aqueous phase. 2 volumes of ice-cold 100%Ethanol is added and
mix well by inversion. It is then centrifuge at approximately 4,300 X g for 15 min at room
temperature and decant the supernatant

9. 1 volume of 70% Ethanol is added and mix well by inversion to the RNA pellet. It is then
centrifuge at approximately 4,300 X g for 15 min at room temperature and decant the
supernatant

10. Excess ethanol is removed with a Pasteur pipette allowing the Ethanol to slowly enter
the pipette by capillary action

11. RNA pellet is allowed to air dry for a few minutes. RNA pellet is then dissolved in an
appropriate volume of TE Buffer which is usually around 100 μl per 1 ml of whole blood.

Determine the integrity, purity and concentration of sample

The most common method used to assess the integrity of total RNA is to run an aliquot
of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). The
secondary structure of RNA alters its migration pattern in native gels so that it will not migrate
according to its true size. Intact total RNA run on a denaturing gel will have sharp, clear 28S and
18S rRNA bands. The 28S rRNA band should be approximately twice as intense as the 18S rRNA
band. The 28S rRNA band should be approximately twice as intense as the 18S rRNA band.
Completely degraded RNA will appear as a very low molecular weight smear.
References:

1. Agilent. (n.d.). Total RNA Definition | Advanced Analytical (AATI). Retrieved April 24, 2019,
from https://www.aati-us.com/define/Total RNA/.

2. Purification of High-quality RNA from Blood Samples with the Thermo Scientific King Fisher
Pure RNA Blood Kit. (2013). Bio Techniques, 55(5).doi: 10.2144/000114105.

3. Sambrook, J. and Russell, D.W.S., 2006. The condensed protocols from molecular cloning: a
laboratory manual (No. Sirsi) i9780879697723).

4.