Lab 1 15/2/2023

RNA Extraction
•Aim
Isolation of clean and intact RNA.

•Principle
Tri-RNA Reagent is a reagent from the improved phenol and guanidine
isothiocyanate (GSN) where Guanidinium isothiocyanate (powerful protein
denaturant) responsible for inactivation of RNAses and Acidic phenol/chloroform
responsible for partitioning of RNA into aqueous supernatant for separation.
During sample homogenization or lysis, TRIZOL Reagent maintains the integrity
of the RNA, while disrupting cells and dissolving cell components. Addition of
chloroform followed by centrifugation separates the solution into an aqueous phase
and an organic phase. RNA remains exclusively in the aqueous phase. After
transfer of the aqueous phase, the RNA is recovered by precipitation with
isopropyl alcohol. After removal of the aqueous phase, the DNA and proteins in
the sample can be recovered by sequential precipitation. Precipitation with ethanol
yields DNA from the interphase, and an additional precipitation with isopropyl
alcohol yields proteins from the organic phase. Co-purification of the DNA may be
useful for normalizing RNA yields from sample to sample.

•Materials
Item Description
Plastic wares Rack, waste container, eppendorfs, tips, micropipettes, ice box
Glass wares Beaker
Other tools Marker, lab coat, gloves, stop watch
Equipment -Cooling centrifuge [Serial no. 179555 – model 3-14KS. Sigma company]
-Thermal block [Serial no. 2B0533014 – type TB1. Biometra company]
-Gel electrophoresis system [Serial no. 2403301. BioRAD company]
-UV Spectrophotometer
-Refrigerator [Serial no. 179555. White Whale]

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 -Vortex mixer [Serial no. B091100654. Grant-Bio PV-1 company]

Reagent Uses
DEPC (Diethylpyrocarbonate) -An alkylating reagent that binds to the histidine residues
of the catalytic site of the RNases, destroying their
enzymatic activity, and produces RNase-free solutions.
-DEPC water is responsible for deactivation of RNases to
maintain the integrity of RNA during extraction.
RNAse inhibitors Protects RNA from degradation during isolation and
purification and in all downstream applications such as
reverse transcription into cDNA by RT-PCR.
Ethanol or Isopropanol Storage of RNA.
RNase-free buffer Re-suspension of RNA.
Guanidinium isothiocyanate Powerful protein denaturant used during cell lysis
because of its ability to irreversibly inactivate degrading
nucleases (RNases).
Acidic phenol/chloroform Partitioning of RNA into aqueous phase for separation.
TRIZOL reagent Maintains the integrity of the RNA, while disrupting cells
and dissolving cell components.
Isopropyl alcohol -RNA recovery by precipitation.
-Yields proteins from organic phase.
Ethanol -Yields DNA from the interphase by precipitation.
•75% ethanol for washing.
Ice cold PBS Isotonic buffer solution help in getting rid of cell debris
and medium based contaminants without decreasing RNA
purity.
β-meracptoethanol -Reduce disulfide bonds and act as a biological
antioxidant by scavenging hydroxyl radicals.
-Reducing agent that disrupts tertiary and quaternary
protein structures and unfold native proteins.
10X MOPS buffer •200 mM MOPS pH=7.
•50 mM Na-acetate.
•10 mM EDTA pH=8.
-Maintain pH levels, inactivate RNase and enzymatic
activity.
Sample buffer •200 µl 10x MOPS buffer.
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10X loading buffer
Electrophoresis buffer
Formaldehyde
Formamide
Glycerol in loading dye
Bromophenol Blue in loading dye Color tracker or marker to monitor the end of
•330 µl 37% formaldehyde.
•1000 µl formamide.
•2 µl 0.5 M EDTA pH=8 (final conc 1mM).
•500 µl glycerol (50%).
•475 µl DEPC treated water.
•25 µl (10 mg/ml) ETH. BR (final conc 0.25 mg/ml).
•Few crystals of Bromophenol Blue.
•5x MOPS buffer 300ml.
•37% formaldehyde 44.6ml.
•Distilled water up to 1.5 liter.
-Disrupt secondary structure of RNA.
-Maintain RNA in the denaturation state.
-Protect RNA from RNases.
Act as solubilizing agent and RNA denaturing agent.
Increase sample density to help the sample to sink in the
gel during the run.
electrophoresis run.

•Methods “TRIZOL tissue RNA extraction, Mice Liver Protocol”

Group A samples: [Female mice: muscles, ovaries]
•2C1 “2 Control sample muscles”
•I2 “Induced group ovaries”
•Co2 “Compared group ovaries”

-Protocol steps:
1) Weigh fresh tissue sample (50-100 mg recommended) and transfer to a 5 mL
micro-centrifuge tube.
Samples should be stored at -80°C until used and kept on dry ice during extraction until TRIZOL
reagent is added.

2) Place a homogenization Teflon probe into the homogenizer and wash the probe
in 70% ETOH followed by 2 waters.
When using the homogenizer, move up in speed slowly and try not to use speeds higher than 3.

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3) Place tube with sample on ice and add 1 ml of TRIZOL reagent into the tube.
The sample volume should not exceed 10% of the volume of TRIZOL Reagent
used for the homogenization.

4) Homogenize tissue on ice by placing the lower portion of the homogenizer

probe in TRIZOL solution until no visible particles remain.
Incomplete homogenization results in significantly reduced RNA yields. Keep homogenized
samples on ice.

5) Take sample out of the ice and incubate at room temperature for 5 minutes.
During this 5 minute incubation, transfer the homogenate into a 1.5 ml labeled
micro-centrifuge tube (1 ml each).

6) After the incubation, add 200 μl chloroform to each sample, shake vigorously by
hand for 15 seconds and incubate at room temperature for 2-3 minutes.

7) Centrifuge at 10,000 rpm for 15 minutes at 4°C.

The mixture separates into a lower phenol-chloroform phase, an interphase, and a colorless
upper aqueous phase. RNA remains in the aqueous phase. The upper aqueous phase is ~50% of
the total volume.

8) Transfer the aqueous phase into a new labeled 1.5 ml micro-centrifuge tube (by
angling the tube at 45°) and pipetting the solution out.
Avoid drawing any of the interphase or organic layer into the pipette when removing the
aqueous phase.

9. Add equal vol. of 100% isopropanol to the aqueous phase (0.5 ml of isopropyl
alcohol per 1 ml of TRIZOL Reagent), and allow it to stand 10 min. (at room temp).

10) Centrifuge at 10,000 rpm for 10 minutes at 4°C.

RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom
of the tube.

11) Remove supernatant then add 1 ml 70% (ice cold) ethanol to RNA pellets.
Then centrifuge (10.000 rpm) 5 min. at 4ºC.

12) Pour off the supernatant.

If the pellet is visible, pipette off any excess ethanol & let air dry for 10-15 min. Pellet should be
almost completely dry, but a little moisture will help it dissolve better.

13) Re-suspend the RNA pellet in 30 μl DEPC treated water (RNase-free water).

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30 μl if the pellet size is small, and 50 μl if the pellet size is large. Leave on ice for 10-15 minutes
and then mix by passing the solution up and down through a pipette tip.

14) Measure RNA absorbance at 260 and 280 nm by diluting DNA extract 1:100
with distilled H2O (total vol. 500 μl).

15) Calculate RNA concentration and comment on the purity.

16) Prepare the sample for the gel. [5 μl dye : 5 μl DEPC water : 5 μl sample]

17) Check for integrity and purity by running a sample on native agarose gel.

-Native Agarose Gel Electrophoresis preparation:

•Sample preparation:
Mix the following in an eppendorf:
5 μl sample (containing 5 – 15 μg RNA)
3 μl Sample loading buffer

•Electrophoresis:
1) Place the solidified gel in its place in the electrophoresis tank.
2) Fill in the electrophoresis tank with electrophoresis buffer.
3) Load the prepared RNA samples.
4) Plug in the electrodes and apply 5-10 V/cm of gel (i.e. 50 – 100 volt for 10 cm
long gel).
5) After the blue front reaches close to gel end, switch off the volt and examine the
gel on a transilluminator.

-Calculations:
Purity of RNA absorbance at 260 and 280 nm = 1.8-2

1C1 at 260 = 0.062

1C1 at 280 = 0.031

Concentration =
A 260∗Dilution factor∗40 ( μmlg ) = 0.062∗1000∗40
1000
= 2.48 μg/μl
1000

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 A 260 0.062
Purity = A 280 = 0.031 = 2 “Good purity”

2C1 at 260 = 0.035

2C1 at 280 = 0.023

0.035∗1000∗40
Concentration = 1000
= 1.4 μg/μl

0.035
Purity = 0.023 = 1.52 “Sample may have organic solvents as phenol and
chloroform”

I2 at 260 = 0.038

I2 at 280 = 0.018
0.038∗1000∗40
Concentration = 1000
=1.52 μg/μl

0.038
Purity = 0.018 = 2.1 “Sample may contain DNA contamination”

Co2 at 260 = 0.090

Co2 at 280 = 0.048

0.09∗1000∗40
Concentration = 1000
= 3.6 μg/μl

0.09
Purity = 0.048 = 1.875 “Good purity”

•Expected results
-Purity within 1.8-2 range.

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 •Real results
Sample A260 A280 Purity Conc of RNA Volume will be
taken for cDNA
1C1 0.062 0.031 2 2.48 2
2C1 0.035 0.023 1.52 1.4 3.5
I2 0.038 0.018 2.1 1.52 3.5
CO2 0.090 0.048 1.845 3.6 1.5

•Discussion
-1C1: good purity because it is in the normal range.
-2C1: may be the sample contain residual organic solvents
(phenol/chloroform) in RNA.
-I2: fairly good but may contain nucleic acids contaminants.
-Co2: good purity because it is in the normal range.

•Trouble shooting
Problem Possible reasons Possible solutions
Low yield of RNA -RNA not solubilized -For solubilization:
completely. •Pippet RNA pellet in SDS or
-Sample not homogenized DEPC-treated water.
completely. •Heat sample at 55º C for 10-15
min.
•Don’t allow RNA pellet to dry
completely.
-For homogenization:
•Make sure to incubate for 5 min
at RT after homogenization.
•Make sure no particulate matter

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 remains.
Degraded RNA -Improper storage of RNA. -Store isolated RNA at -70º C not
-Sample manipulated too much -20º C.
before the addition of TRIZOL -Process tissue immediately after
reagent. removal from animal.
-Frozen tissue thawed in -Minimize washing steps for cell
absence of TRIZOL reagent. culture samples.
-Add TRIZOL reagent directly to
plates. Don’t trypsinize cells.
-Add frozen tissue to TRIZOL
reagent.
Low A260/280 -Organic solvents “phenol- -Precipitate RNA again with
(<1.65) chloroform” in RNA. ethanol.
-Sample not homogenized with -Make sure not to carry any of
sufficient TRIZOL reagent. organic phase with RNA sample.
-pH of solution is acidic. -Make sure to incubate sample for
-A260 or A280 outside the 5 min at RT after homogenization.
linear range. -Dissolve sample in TE instead of
water.
-Dilute sample to bring
absorbance into linear range.
RNA contains -Part of interphase was removed -Make sure not to take any of
DNA with aqueous phase. interphase with aqueous phase.
-Insufficient TRIZOL reagent -Make sure that original sample
used. doesn’t contain any organic
-Cells contained organic solvents.
solvents. -Remove any particulate material
-Insoluble materials weren’t before chloroform addition.
removed before chloroform
extraction.

•Conclusion
-Some samples are found to be good to work on (Samples with good purity: 1C1
and CO2).

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