RNA isolation

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Joseph dela Cruz

 

Lab Report #4

1.      Date:                   April 28, 2012

2.      Title:                   RNA Isolation

3.      Purpose:             To understand and be able to isolate RNA for other molecular techniques such as RT-PCR.

4.      Principle:

The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. Trizol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues. Trizol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. RNA remains only in the aqueous phase.      

5.      Materials

Cell culture in 6-well plates

PBS

Trizol

Chloroform

Isopropanol

Ethanol

DEPC-treated water

              

6.      Methods

a.      Wash the cells (6-well plate on ice) twice with PBS. Add 0.5ml Trizol to each well, remove cells with a cell scraper and transfer to a new e-tube.

b.      Add 200µL chloroform, vortex for 30sec and keep on ice for 5mins. Centrifuge at 15000 rpm for 15mins at 4°C. Transfer 400µL of the upper aqueous part to a new e-tube.

c.       Add 400µL of isopropanol, vortex for 30sec and keep on ice for 5mins. Centrifuge at 15000 rpm for 10mins at 4°C. Remove the supernatant.

d.      Add 1ml of cold 75% EtOH for washing, dissolve the pellet by vigorous pipetting and then centrifuge at 15000 rpm for 10mins at 4°C. Remove the supernatant.

e.      Air-dry the RNA pellet (until transparent) at RT for 5mins, add 20µL of DEPC-treated water to completely dissolve the RNA pellet by a pipette. Incubate at 57°C for 10mins.

f.        Measure absorbance at 260-280nm.

g.      Store RNA samples at -70°C or do RNA electrophoresis.

 

7.      Discussion

RNA are being isolated and used because most eukaryotic genes contain introns which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact DNA sequence which would be directly translated into protein after transcription.

 

Any extracted RNA must be devoid of contaminants such as salt, protein, solvents and genomic DNA. Poor quality RNA will lead to problems when performing the reverse transcription and labelling and might affect data quality. Quality control of extracted RNA is done using gel electrophoresis and optical density measurements. Gel electrophoresis is used to check for Genomic DNA contamination and RNA decay. Optical density is used to assay the RNA yield and to check for contamination by salt, solvent, protein, etc.

Common RNA purification methods

Phenol based extraction methods. These single-step RNA isolation methods based on Guanidine isothiocyanate (GITC)/phenol/chloroform extraction are very popular because they require much less time than traditional methods (e.g. CsCl₂ ultracentrifugation). Many commercial reagents (e.g. Trizol, RNAzol, RNAWIZ) are based this principle. The entire procedure can be completed within an hour to produce high yields of total RNA. However, this technique does require the use of toxic chemicals like phenol/chloroform and GITC.

Silica gel - based purification methods. RNeasy is a very popular purification kit marketed by Qiagen. It uses a silica gel-based membrane in a spin-column to selectively bind RNA larger than 200 bases. The method is quick and does not involve the use of phenol. Although the yield from an RNeasy column is usually not as high as phenol extraction method, the entire procedure takes even less time than the phenol based method.

Oligo-dT based affinity purification of mRNA. Due to the low abundance of mRNA in the total pool of cellular RNA, reducing the amount of rRNA and tRNA in a total RNA preparation greatly increases the relative amount of mRNA. The use of oligo-dT affinity chromatography to selectively enrich poly (A)+ RNA has been practiced for over 20 years. The result of the preparation is an enriched mRNA population that has minimal rRNA or other small RNA contamination. mRNA enrichment is essential for construction of cDNA libraries and other applications where intact mRNA is highly desirable. The original method utilized oligo-dT conjugated resin column chromatography and can be time consuming. Recently more convenient formats such as spin-column and magnetic bead based reagent kits have become available.

 

8.      References

http://www.isletbiology.com/SOPs/MB1_1_RNAbasics.pdf

www.ambion.com/techlib/basics/rnaisol/index.html

http://bioquestion.com/protocols/80-rnaextraction.html

http://www.sabiosciences.com/newsletter/RNA.html