isolation of RNA from blood principle protocol and functions of reagents

RNA extraction is simply the process of extraction of purified RNA from the source. The source can be anything blood sample bacterial cell animal cell or plant cell. Extraction of RNA is a comparatively sensitive process because RNA is not as stable as DNA as it is single stranded. So methods of extraction of DNA cannot be applied directly for isolation of RNA because it is much more sensitive. Basic principle is the breakdown of cell wall (in case of bacteria and plant cells) and cell membrane, removal of all other contaminants (protein, lipids, DNA and carbohydrates for eukaryotic cells), concentration of RNA and analysis.

Principle:

principle of RNA extraction from blood sample

Fig.01: principle of RNA extraction from blood sample

The key to success in RNA extraction is speed. Cellular RNases should be removed and inactivated as quickly and early as possible during first few steps of extraction. Once RNases are inactivated less is the threat to the integrity of the RNA and purification can be proceeded at more graceful pace.  First cells are lysed by the breakdown of cell wall in case of plat and bacterial cell followed by break down of cell membrane. All the internal components are exposed including RNA. The resulted fluid is called lysate. Cell lysis can be achieved by using buffers and other reagents like guanidinium isothiocynate, guanidinium chloride, sodium dodecyl sulphate (SDS), sarcosyl, urea, phenol or chloroform.Different types of buffers and chemicals are used are available to do the cell lysis but for RNA preferably TRIZOLE reagent is used. It is ready to use reagent which maintains the integrity of RNA during homogenization and at the same time disrupts the other cellular components. Now our goal is to remove all contaminates from the cell lysate to obtain purified RNA.  Lipids and proteins are removed by addition of chloroform which gives us two layers aqueous layer and organic layer. RNA remain in organic layer. DNase can be added to remove the DNA and Proteinase k can be added for removal of protein. Alternatively repeated organic extraction can also be done by using chloroform and phenol for the removal of proteins. RNase inactivation is the most important and crucial step of RNA extraction because RNases are abundant in environment and it can be done by using chloroform and phenol. RNA can be separate from the cellular components by using organic extraction followed by centrifugation giving RNA in aqueous phase and cell debris in the organic phase. Concentration of RNA can b done by addition of isopropanol.

phase separation method
Fig.02: phase separation method

Trizole:

TRI in trizole stands for total RNA isolation. It is a ready to use regent contain phenol, guanidinium salts, red dye and other components for the isolation of RNA in a single step process. Trizole was developed by Piotr chomczynski and sacchi in 1987. Red dye present in the reagent is used for easy detection of organic phase and do not interacts with RNA.

working of trizole

Fig.03: working of trizole

Physical properties of Trizole:

PH of 10 x dilution 4.8-4.9
Boiling point 181.8 degree
Melting point 40.85 degree
Specific gravity 1.058

Guanidinium salts:

Trizole is basically solution of phenol and guanidinium salts which ensure the single step isolation of RNA so these salts are very important in reagent. Guanidinium salts (guanidinium hydorochloride or guanidinium thiocynate) disrupts the cells, solubilize their components and denature the endogenous RNases. These are chaotropic agents that destroy 3D structure of protein. Above mention salts convert proteins to randomly collide state. The mechanism by which exactly this denaturation is done still unclear, though it seems to involve binding of greater amounts of guanidinium salts to protein as the denaturation proceeds. Gaunidinium chloride was used first time for the isolation of RNA.

Though guanidinuim chloride is strong inhibitors of ribonuclease but it is not powerful enough denaturant to allow extraction of RNA from tissues which are rich in RNase like pancreas. Guanidinium thiocynate is strong chaotropic agent containing active anionic and cationic groups that are able to form strong hydrogen bonds. It breaks the disulfide bonds of protein in the presence of any detergent which is used to break hydrophobic interactions.

Structure of guanidinium hydrochloride:

HN=C (NH2)2.HCl

Structure of guanidinium isothiocyanate:

HN=C (NH2)2.HSCN

How to win battle against RNases ?

Isolation of RNAi s very sensitive and difficult process because of RNases as they are present everywhere in the environment even they are released from the pores of fingers, many experiments are needlessly ruined by the contamination of RNase.

Activity of RNases

Fig.04: Activity of RNases (a) cleavage of probe due to RNases (b) control reaction without RNases

Following are the two main reasons of contamination with exogenous RNase

  • Contaminated buffers
  • Automatic pipetting

Contaminated buffers:

Due to poor aseptic technique buffers may become contaminated with bacteria or any other microorganism. The growth of microorganism is not visible with naked eye and need to be florid to cause any problem. RNase cannot be removed by autoclaving the solutions so they must be discarded

Automatic pipetting devices:

There is simply no point in using disposable pipette tips that are free of RNase if the automatic pipettor has been previously used to dispense the solution containing RNase. If the metal ejector of pipettor cones in contact with the sides of tubes it becomes vector for the dissemination of RNase.

Inhibitors of RNase:

RNases are powerful enough to degrade the RNA at any stage of isolation or characterization. Therefore it is necessary to use RNase inhibitors. Some commonly used inhibitors are

  • Diethylpyrocarbonate (DEPC)
  • Vanadyl ribonucleoside complexes
  • Protein inhibitors of RNases

Diethylpyrocarbonate (DEPC):

It is a highly active alkylating agent which is used to inhibit the activity of RNases in buffers or glassware.It is used in molecular cloning to inactive the minute amount of RNases that may contaminate solution, glassware or plastic are used during process of RNA isolation. DEPC is highly active and can destroy the active site of RNases by ethoxyformylation of histidyl groups.

In aqueous solution, DEPC hydrolyzes to CO2 and ethanol, having half-life of ~20 minutes in phosphate buffer (pH=6.0) and ~10 minutes at pH 7.0. This process is greatly accelerated by Tris and other amines that is why DEPC is not used in solutions that contain these buffers. DEPC that are free of nucleophiles are highly stable but minute amounts of these solvents can cause conversion of DEPC to diethlycarbonate. DEPC should also be protected against moisture so it should be stored in dry conditions and bottle should be at ambient temperature before opening the bottle. Although purified water by modern reverse osmosis system is free of RNases, poorly maintained system may become contaminated by microbial contamination. In such cases it is advised to use DEPC-treated water by treating the water with 0.1% DEPC for 1 hour at 37 degrees and should be autoclaved for 15 minutes at 15psi.

Draw backs of DEPC:

It may degrade to CO2 and ethanol which increases the ionic strength of unbuffered solutions. DEPC can also cause the carboxymethylation of unpaired adenine residue in RNA .mRNAs that have been exposed to DEPC are translated with reduced efficiency in vitro protein- synthesizing systems.

Vanadyl ribonucleoside complexes:

These are transition state analogues that have ability to bind the active site of RNases and inhibit their catalytic activity.They do not bind to RNases covalently hey must be used all stages of RNA extraction and purification. However these complexes inhibit RNA polymerase and invitro translation, they must be removed from final preparation of RN by multiple extractions with phenol containing -0.1% hydroxyquinoline.

Protein inhibitors of RNases:

RNases have ability to bind very tightly, non-covalently to 50-kD proteins found in the cytoplasm of all mailman tissues and can be isolated from placenta. The archetypal RNase inhibitor is horseshoe-shaped molecule, containing 7 leucine rich repeats, 28 and 29 residues in length.

Ribonuclease inhibitor showing its horse-shoe shape

Fig.05: Ribonuclease inhibitor showing its horse-shoe shape

It also contain large number of reduced cysteinyl residues. Because they do not form covalent complexes that is why they cannot be used in the presence of denaturants like SDS and guanidine. These inhibitors must be replenished several time during the process of extraction of RNA as they are removed by extraction with phenol.

Is your RNA intact? Methods to check RNA integrity:

Regardless of downstream applications it is better idea to check integrity of RNA. Most common method to check the integrity of RNA is agarose gel electrophoresis with ethidium bromide dye. Intact RNA run on gel should have 28s and 18s rRNA bands. 28s rRNA should be twice as intense as 18srRNA band. This 2:1 is best indication the intact RNA is isolated.

RNA integrity

Fig.06: Intact vs. Degraded RNA. Two µg of degraded total RNA and intact total RNA.  The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample. The degraded RNA appears as a lower molecular weight smear.

 Degraded RNA may give smear type appearance without sharp bands of rRNA or they will not exhibit 2:1 or 28s rRNA and 18s rRNA. A drawback of using agarose gel is the amount of RNA for integrity check, generally 200ng should be added in well in order to visualize with ethidium bromide as in some RNA preparations e.g. needle biopsies or laser capture microdissected sample gives very low yields of RNA.

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