Isolation and Estimate the Total RNA Content from Plant Tissues

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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  Most plant material contains relatively high levels of RNAse activity mostly located in the vacuoles. During the plant RNA extraction it is necessary to completely break cells, minimise the activity of RNAse released during cell lysis and avoid the accidental introduction of the minimum trace of RNAse from any other source in the laboratory.²  RNAse is highly regulated in cell metabolism, but when cells are lysed the regulatory mechanisms are disrupted. Plants contain hundreds of copies of Rrna genes for cytoplasmic ribosomes, mitochondrial ribosomes and chloroplast ribosomes.³  Large and small rRNA molecules of 3 different origins possess different molecular weight which can be further separated and identified by agarose gel electrophoresis.

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

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REQUIREMENTS

Materials:        Aagarose

          TSB Buffer (100 mM Tris-HCL containing 1% SDS & 2% 2-mercaptoethanol, pH 7.5)

          Acid Phenol [APC] (pH 4.3)

          Chloroform for analysis

         2-Propanol for analysis

         70% ethanol

         3M ammonium acetate

     8M LiCl

    10 mg/ml ethidium bromide (highly toxic)

    Running buffer (1x TAE- 2M tris, 1M acetic acid, 50 mM EDTA; 2% formaldehyde)

    FFM (50% formamide, 6% formaldehyde, 1x TAE, 0.25% bromophenol)

    50x TAE

Apparatus:     Mortal & Pestle

        1.5 ml disposable tubes

        Pipettors

        Disposable tips

        Gloves

        UV Spectrophotometer

        Quartz cuvettes

        Vortex

        Microcentrifuge

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PROCEDURE

Harvest 5 g of spinach leaves and grind with liquid nitrogen in a mortar pestle to a fine powder. Distribute 0.5 g of powder into Eppendorf tubes and then add 250 µl of APC and TSB buffer. Vortex until a complete emulsion is formed. Centrifuge it at 13000 rpm for 2 min. Transfer the aqueous phase to a new tube. Add 500 µl of APC and the centrifuge at 13000 rpm for 2 min.

Again take the upper phase and then add 500 µl of chloroform and again centrifuge.⁴ 
Collect the upper phase; add 50 µl of 3M ammonium acetate and mix. Add 500 µl of 2-propanol and precipitate for 15 min on ice. Then centrifuge at 13000 rpm for 10min as a result of which a white pellet is visible. Remove the supernatant and rinse with 500 µl of 70% ethanol. Centrifuge at 13000 rpm for 2 min.

Remove the supernatant and dry. Dissolve the pellet in 210 µl distilled water. Add 70 µl of 8M LiCl and place on ice for 30 min. Centrifuge for 15 min at 13000 rpm. Remove the supernatant and dry. Dissolve the pellet in 100 µl of distilled water. Prepare 2 dilutions of 10 µl & 20 µl of RNA in 490 µl & 480 µl distilled water.

Read the absorbance at 260 nm to calculate the RNA concentration in both dilutions. Also read the absorbance at 230 & 280 nm to calculate the ratios A₂₆₀/ A₂₃₀ & A₂₆₀/A₂₈₀ to check the purity. Add 10 µl FFM to each 10 µl RNA. Then boil for 5 min to denature the RNA.For agarose gel electrophoresis a 7×10 cm tray is used where 1.5 mm thick 8 well combs are used.⁵  To melt the agarose heat 50 ml of 1.5% agarose in 1X TAE. After cooling to 50^oC add 2.5 µl of ethidium bromide solution.

Prepare 250 ml of running buffer in formaldehyde. Pour the agarose and allow it to solidify. Remove the gel and place in apparatus. Load the samples & carry electrophoresis for 1-2 hr. When the dye front has migrated 2/3^rd of the way, stop the electrophoresis and place the gel on a UV light box & take a photo. Care should be taken while seeing through a UV light. Make sure to wear a full face mask to prevent UV damage to eyes & face.

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CONCLUSION

RNA content of any plant tissue can be estimated by this procedure.

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REFERENCES

1. Butler J.N., Peirson S.N. RNA extraction from mammalian tissues. Methods in molecular biology. 2007: 362:315-27
   
2. Spindler S., Siebert P., Jurnak F., Coffman F., “Isolation of biologically active mRNA”. Practical biochemistry for colleges-Pergamon Press, Oxford: 1989: 143-145.
   
3. Marano M.R., Orellano E.G., Carrillo N., “Practical exercise involving RNA isolation”. Biochem Edu: 1994: 207-210.
   
4. Doods J.A., Tremaine J.H., Ronald W.P., “some properties of carnalion ringspol virus single and double stranded RNA” . Virology: 1977: 83; 322-328.
   
5. Claros M.G., Canovas F.M., “Rapid high quality RNA preparation from pine seedlings”. Plant Molecular Biology. 1998: 16; 9-18.