Isolation and Estimate the Total RNA Content from Bacterial Cells

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BACKGROUND

RNA abbreviation is ribonucleic acid is a complex compound of high molecular weight that functions in cellular protein synthesis and replaces DNA as a carrier of genetic codes in some viruses.¹ RNA is a polymeric molecule that is essential in various biological roles in coding, decoding, regulation and expression of genes. RNA is a nucleic acid and along with lipids, proteins and carbohydrates constitute the major macromolecules essential for all known forms of life. RNA molecules are single-stranded nucleic acids composed of nucleotides.² RNA plays a major role in protein synthesis as it is involved in the transcription, decoding and translation of the genetic code to produce proteins. RNA stands for ribonucleic acid. RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached by phosphodiester bonds, forming strands of varying lengths.

So the basic objective of the test is to isolate and estimate the total RNA content from bacterial cells.

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REQUIREMENTS

Materials:    Buffer A:   Buffer A saturated phenol (1.2 ml per sample)

                 Phenol- Chloroform (0.6 ml per sample)

                 3 M NaOAc (pH 5.2) (90 µl per sample)

                 DEPC treated dH₂O (1.5 ml per sample)

                 Absolute ethanol (2 ml per sample)

                70% ethanol (1 ml per sample)    

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PROCEDURE

Extraction

The tubes are removed from the -70^oC and then immediately 500µl of complete Buffer A. The tubes are vortexed to resuspend the cells. The tubes are centrifuged in a micro centrifuge for 30 sec at full speed. The layers are removed using an RNase free blue tip. Then 600 µl of Buffer A is again added. The tubes are again centrifuged in a micro centrifuge for 2-3 minutes at full speed. The aqueous layer is removed to a new tube. Then 600 µl of 1:1 phenol buffered with chloroform at room temperature. The samples are mixed by vortexing for 20 seconds. Separate the layers by centrifuging the tubes in a micro centrifuge for 2-3 minutes at full speed. The aqueous layer (top layer) was removed to a new tube. 50 µl of 3M NaOAc (pH 5.2) and 1ml absolute ethanol. The pellets are resuspended in 400 µl of dH₂O. The pellets are washed by adding 1ml of 70% ethanol and vortexing for 20 seconds. The micro centrifuge tubes are centrifuged at full speed for 5 minutes. The supernatant is then removed. The open tube is then incubated at 37^oC for 5 minutes to dry the pellet. The RNA is dissolved in 50 µl dH₂O. It is then vortexed and centrifuged briefly. 5 µl RNA is diluted into 495 µl of dH₂O.³

Isolation

The method of isolation is known as guanidium thiocyanate phenol chloroform extraction. TRIZOL is light sensitive and is often stored in a dark coloured, glass container covered in foil. It must be kept below room temperature.⁴ When used, it resembles cough syrup, bright pink. The smell of the phenol is extremely strong. Caution should be taken while using TRIZOL as exposure to TRIZOL can be a serious health hazard. Exposure can lead to serious chemical burns.⁵ So lab coat and gloves are strictly recommended.

1 ml of TRIZOL is added to sample and homogenized. 200 µl of chloroform is then added to homogenate and vortexed. It was then centrifuged at 12000rpm for 15 min. The aqueous layer is transferred to fresh tube. The RNA is precipitated by mixing with 0.5 µl Isopropanol. Then centrifugation is repeated at 12000 rpm for 10 min and then the supernatant is removed. The pellet is washed with 1 ml of 70% ethanol. Then centrifugation is repeated at 7500 rpm for 10 min and then the supernatant is removed & air dried. The RNA pellet is dissolved in appropriate volume of RNase free H₂O.⁶

Other methods of extraction⁷

Organic extraction methods

In this process the sample is homogenized in a phenol containing solution and the sample is then centrifuged. During centrifugation the sample separates into 3 phases: a lower organic phase, middle phase that contains denatured proteins & an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation.

Filter based RNA isolation

This method utilizes membranes that are seated at the bottom of a small plastic basket. Samples are lysed in a buffer that contains RNase inhibitors then are bound to the membrane by passing the lysate through the membrane using centrifugal force. The solutions are passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation.

Magnetic Particle method

This method utilizes small particles that contain a paramagnetic core. Paramagnetic particles migrate when exposed to a magnetic field but retain minimal magnetic memory once the field is removed. This then allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field and then be resuspended easily once the field is removed. Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles are collected by applying a magnetic field.

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CONCLUSION

By the above procedure we can isolate and estimate the total RNA content from bacterial cells.

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REFERENCES

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3. Bird M. “Extraction of RNA from cells and tissues”. Methods Mol. Med. 2005- 108: 139-48
   
4. Harder J., “RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin”. Journal of biological chemistry. 2002- 277(48): 46779-46784.
   
5. Chomczynski P., Sacchi N., “The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on”. Nat Protoc. 2006: 1(2): 581-5
   
6. Luhtala N., Parker R.,” T2 family ribonucleases: ancient enzymes with diverse roles”. Trends in biochemical sciences. 2010: 35(5): 253-259
   
7. Dyer K.D., Rosenberg H.F., “The RNase a superfamily: Generation of diversity and innate host defense”. Molecular Diversity. 2006- 10(4): 585-597