TRIzol RNA Extraction Principle ,Protocol ,Functions of Reagents

Introduction

RNA extraction is the isolation of total RNA present in
plant, animal, yeast or bacterial cells. There are quite a lot of methods to
extract the RNA from various cells but RNA isolation by TRIzol Reagent is the precisely
stress-free and gives high RNA yield.
By using this method, you can isolate RNA, DNA as well as protein from the same
biological sample. TRIzol reagent effectively inhibits the RNase activity
thereby maintaining the RNA integrity. It also facilitates the solubility of
cell components while disrupting the cells to extract RNA. It takes almost
60min to isolate the RNA by using TRIzol reagent. RNA is extracted for various
biological purposes such as Reverse transcription polymerase chain
reaction (RT-PCR), Dot Blot hybridization, poly (A) + selection, Northern
Blot analysis, RNase protection assay and molecular cloning.

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Principle

First of all the biological sample is lysed accordingly be
it bacterial, plant or animal cells by using ice-chilled TRIzol Reagent. The
broken cells are homogenized by pipetting up and down many times. After
transferring the homogenized and broken cells into Eppendorf, Chloroform is
added to the lysed cells. It gives you three layers. Upper aqueous layer
contains extracted RNA, an interphase contains DNA, and proteins are dissolved
in bottom red organic layers. For RNA purification, the pH is kept at around 4,
which holds RNA in the aqueous phase preferentially. After centrifugation,
upper aqueous layer is pipetted out carefully and isopropanol is added to
precipitate the RNA.
(Additionally, you can separate the interphase and lower organic phase to
extract the DNA and proteins respectively. DNA is precipitated by ethanol and
proteins are precipitated by isopropanol). Again precipitated RNA is centrifuged
to get RNA pellet which is washed with 70% ethanol (made with DEPC treated
water) , air-dried and suspended in DEPC treated (RNase free) water. Extracted RNA
is mostly quantified to calculate the yield by Picodrop Microliter
Spectrophotometer. It can be stored at -80^oC or converted into cDNA
and stored at -20^oC for subsequent applications.

Functions of reagents

1. TRIzol Reagent

TRIzol Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components (isoamyl alcohol) which facilitate the extraction of a variety of RNA species of large or small molecular size. It has acidic pH so that the RNA, DNA and proteins get separated in 3 different phases, upper aqueous, interphase and lower organic phase. TRIzol mostly contains the following components.

a) Guanidine Isothiocyanate :This is the component which lyse the cells to extract RNA. It acts as chaotropic agent, hence, maintaining the integrity of RNA by breaking the hydrogen bonding between water molecules and weakening the hydrophobic effect on RNA. It also aids denaturation of RNase.
b) Phenol: phenolic component of TRIzol reagent helps to dissolve the broken proteins and RNases in itself. The lower red organic phase after adding isopropanol is mostly phenol.
C) Isoamyl Alcohol: It works as anti-foaming agent and sharpens the boundary of aqueous and organic phase. It also ensures the deactivation of the RNases. Otherwise RNase can degrade the extracted RNA.

2. Isopropanol (2-propanol)

RNA is insoluble in isopropanol. Therefore, it helps to precipitate the RNA which is obtained in the form of pellet after centrifugation.

3. Ethanol

The RNA pellet is washed with 75% ethanol which dissolves all the organic impurities present in the RNA pellet. It evaporates quickly so avoid over drying the RNA pellet.

4. RNase Free Water:

Water is treated with Diethyl pyrocarbonate (DEPC) which covalently modifies the histidine, lysine,cysteine and tyrosine residues of RNase and deactivates it. RNA pellet is resuspended in DEPC treated RNase free water to be stored at -80^oC.

Protocol

Lysis of Biological Sample

The method of lysing the cells depends upon the cell type. The type biological Sample and its way to lyse the cells to extract RNA is given below.

Sample Type	Amount of the Sample	TRIzol Reagent	Method to Lyse
Tissue	100mg	1000ul	Take tissue in Eppendorf, add trizol in it, homogenize.
Monolayer of Cells	1 Million Cells	500ul	Remove the growth media from cell culture plate, pour TRIzol directly onto it without washing with Phosphate Buffer Saline (PBS)
Cell Suspension	5 Million Cells	750ul	Get the pellet of the cells by centrifugation in 15ml tube, pour TRIzol in it, homogenize by pipetting up & down many times.

Having poured the TRIzol Reagent, incubate at room
temperature for 5min to completely degrade the protein and cell membranes. After
incubation, the lysed cells are sometimes called as cell lysate. You can store
the cell at -20^OC or -80^OC. Mostly the cell lysate is
stored at this stage because in cell culture lab, RNA is extracted from a
myriads of different sample at picked up at various time points; and when all
period is complete, all the cell lysates are picked up from the fridge and performed
the subsequent steps to isolate the RNA.

200ul of chloroform is added per 1000ul of TRIzol reagent
into the cell lysate. It is then inverted several times. After the inversion, 3
different layers are visible but you need to centrifuge it at 12000g for 15min
at 4 ^OC to make a sharp boundary between the layers.

After that the upper aqueous layer containing extracted RNA
is removed by using 200ul pipette. The Eppendorf is tilted at 45degree
and it is made sure that the tip of the pipette must not touch the interphase.
This is a very crucial step to isolate high quality RNA.

RNA precipitation

Upper aqueous layer is
pipetted into new Eppendorf and 500ul isopropanol is added per 1000ul of TRIzol
used in the beginning to tear up the cells. It is centrifuged for 10min at
12000g at 4 ^OC. When you will pick up the Eppendorf from the
centrifuge, it is highly likely that you see an RNA pellet on the bottom of the
Eppendorf. Sometimes the pellet is extremely vivid which indicates the protein contamination
with our RNA that is not good at all. Dirtier pellet is more visible. On the
other hand, there may not be a VISIBLE pellet but that does not mean RNA is not
extracted or you should stop further steps. Discard the supernatant and proceed
to the following steps.

Washing RNA Pellet

1ml 75% ethanol is poured onto the pellet and vortex it and
centrifuge at 7500g at 4^OC for 5min. (You may also store the DNA at
this stage, but storing the RNA in ethanol is good but recovering the RNA from
it does not give high yield of RNA). Discard the supernatant and let the pellet
of the RNA air dry by placing the Eppendorf’s lid open. It takes 10min to
evaporate all the ethanol. Ovid over drying the pellet.

Store the RNA

Air dried pellet is resuspended in 50ul RNase free water.
Pipette it few time to completely solubilize the RNA into the water and store
it at -80^OC. It is suggested that you must make cDNA from this
extracted RNA and store cDNA which is more stable. Read cDNA synthesis by
RT-PCR.

Quantification of RNA

Before storing the RNA, 5ul of the sample is taken from it, diluted it two times (5ul RNA sample + 5ul DEPC treated water). Absorbance of this sample is taken at 260nm by Picodrop Microliter Spectrophotometer. All you have to do is take 2.5ul of diluted RNA sample and put it into the Picodrop machine. It gives you a value which is the amount of RNA in Nanogram per mL or Picogram per mL accordingly. You have to multiply this by 2 in order to find the amount of RNA in your original sample. You should also note the ratio of absorbance at 260nm/280nm. If it is near 1.8, your sample is pure.

NOTE: Most of the time you have to pick up 1000ng or 1ug RNA sample for cDNA synthesis or to run on denaturing gel. So, you have to pick a specific volume of you RNA sample which contains 1ug of RNA. Here is how you calculate this by unitary method.
Suppose your concentration of RNA is 300ng/mL.
300ng is in 1mL
1ng is in 1mL/300
1000ng (1ug) is in 1mL/300 x 1000 = 3.33ul (this means 3.33ul of your original sample will have 1ug of RNA)

Precautions

1. Wear gloves all the time as our skin contains a huge amount of RNases which can degrade our RNA. If you are not good with handling try changing the gloves more often.
2. Always use DEPC treated water or RNase free water for making 75% ethanol, or for making agarose gel. NEVER use tap water or distilled water.
3. You must use RNase free tubes. It is mostly labelled on the packet by the company. If you don’t have RNase free Eppendorfs you must treat them with DEPC.
4. Design your experiment on the note book before actually going to perform it. It will decrease the risk of low RNA yield.
5. Check the availability of the reagents before starting the experiment.
6. Preferentially, you should make cDNA from extracted RNA on the same day.
7. After reading and understanding this whole protocol you can make a flow chart and paste it onto the wall of your lab.

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