BBT413L Plant biochemistry & biotechnology lab

Experiment 2:
Isolation, quantification
and purity determination
of plant RNA using TRIzol
 •RNA (ribonucleic acid) transfer genetic
information in living organisms.

•Found in the nucleus and the cytoplasm

What is RNA? •Key to information flow within a cell

•Different types
RNA
• Messenger RNA (mRNA): 1-5% • sRNA = small RNA
• Serves as a template for protein • siRNA = small interfering
synthesis RNA
• miRNA = microRNA
• • Ribosomal RNA (rRNA): >80%
• Structural component of ribosomes

• • Transfer RNA (tRNA): 10-15%

• Translates mRNA information into the
appropriate amino acid
 Further investigations

– Northern analysis

– cDNA library construction

Why we do the – RT-PCR
extraction?
– Microarray analysis
 RNA
• Presence throughout the cell
purification
benefits
 Due to the presence of –OH group in the ribose sugar, it is more active and is
not stable in alkaline conditions. RNA is more easily subject to attack by
enzymes.
RNA is single stranded, hence its nitrogenous base is not away from other
molecules to react, and thus is reactive.

After transcription, RNAs require proper maturation through a multi-step

RNA process integrating capping, splicing, and polyadenylation. Hence stability a
matter to concern.
purification RNA molecules are variable in length, but much shorter than long DNA
polymers. A large RNA molecule might only be a few thousand base pairs long.
challenges Separating RNA from other cellular components such as proteins, lipids,
pigment etc. Plants pose additional challenges due to the presence of secondary
metabolites, polyphenols and polysaccharides.
Avoiding fragmentation of the RNA molecules by mechanical shearing or the
action of endogenous nucleases.

Avoiding contamination and preservation.

The problem(s) with RNA

• RNA is chemically unstable -- spontaneous cleavage of phosphodiester

backbone
• RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)
• RNases are released upon cell lysis
• RNases are present on the skin
• RNases are very difficult to inactivate
• -- disulfide bridges conferring stability
• -- no requirement for divalent cations for activity
RNase
• Most plant material contains relatively high levels of RNAse activity mostly
located in the vacuoles.
• (i) completely break cells
• (ii) minimize the activity of RNAses released during cell lysis
• (iii) avoid the accidental introduction of the minimum trace of RNAses from
any other source in the laboratory. (autoclave)
• RNAses are highly regulated in cell metabolism, but when cells are lysed, the
regulatory mechanisms are disrupted
• RNAses are not denatured by boiling since the enzyme spontaneously refolds
into its active conformation once the temperature is lowered.
Top 10 sources of RNAse contamination
• Ungloved hands
• Tips and tubes
• Water and buffers
• Lab surfaces
• Endogenous cellular RNAses
• RNA samples
• Plasmid preps
• RNA storage
• Chemical nucleases
• Enzyme preparations
 How to avoid Rnase- Autoclave…..
• Contaminated equipment
• Contaminated solutions/buffers
• Use “RNA-Only” pipets, glassware
• Bake glassware, 300o C, 4 hours
• Use good sterile technique • Use “Rnase-free” pipet tips
• Treat solutions with • Treat equipment with DEPC
DEPC(when possible)
• Make small batches of solutions
The Plant Cell
Two basic approaches
 Organic extraction-how it works

Phenol/chloroform removes protein, lipids and DNA from the

RNA sample and then RNA is separated by ethanol

Advantages Disadvantages
• Can be used for large or small • Use of hazardous chemicals
sample
• Can modify extraction to
remove high level of protein
Column purification-how it works
•Glass filters bind the RNA while other cellular components are
washed away.
•RNA is eluted in a highly purified form

Advantages • Disadvantage
• Rapid procedure • Not as scalable as organic
• No organic solvent required extraction
• No alcohol precipitation
needed
Basic Protocol

Most RNA extraction protocols consist of two parts

1. A technique to lyse the cells gently and

solubilize the RNA along with maintaining the
integrity of RNA
2. Enzymatic or chemical methods to remove
contaminating proteins, DNA, or
macromolecules
Steps in extraction

Homogenization

A.Phase Separation

A.RNA Precipitation

A.RNA Wash
Homogenization
• Break the cells rapidly and efficiently so that the nucleic acids are released from the cells by the
addition of a strong lysis solution.

• Tissue homogenization by using mortar and pestle. Its force and high pressure produce consistent
and uniform samples.

• Lysis solution rapidly disrupts cells without destroying their nucleic acids.
• TRIzol reagent as ‘lysis solution’ combines phenol and chaotropic agents named guanidine
thiocyanate. This Trizol helps to maintain RNA integrity during lysis (one of the best fit denaturant).
• Phenol and chloroform denaturation of DNA and proteins, RNase. Inactivation of RNases also.
 Detergent:
Breaks down the hydrophobic membranes that surround cells
and some cellular organelles.

Lysis
solution
Reductant :
Inactivates RNases.
Preserving RNA during the tissue Chaotrope
disruption process.
Unfolding proteins and other
biomolecules.
Phase separation
• Homogenate (from homogenization step) must be store for 5 min
at RT to permit the complete dissociation of nucleoprotein
complexes.
• Chloroform used to:
• - Separate solution in aqueous phase, interphase and organic
phase
• - RNA in aqueous phase, DNA (interphase) and protein (organic
phase)
• RNA, DNA and protein separation are based on density
centrifugation
• Chloroform is organic solvent. So, lysed cell components that are
hydrophobic will be trapped in these solvent
RNA precipitation
• RNA is often recovered from the aqueous phase using
isopropyl.
• RNA can also be selectively precipitated from DNA
through the use of chloride salts which can selectively
precipitate RNA from DNA as well as proteins
• RNA precipitate (often visible before centrifugation)
forms a gel-like or white-pellet on the side and bottom of
the tube.
RNA wash
• RNA is selectively washed with 75% ethanol since it can wash the RNA
pellet away from any salt residual

• Finally, RNA is resuspended within DEPC mixed water. DEPC itself is a

good RNase inactivator.
RNA isolation
Materials & equipment

Chemicals Sample: Spinach soft leaves

• TRIzol reagent
• 1.2 M NaCl, Equipment (autoclaved)
• Isopropanol • Sterile working bench
• Chloroform • Microfuge tubes
• 75% EtOH • Hand Homogenizer (mortar pestle)
• DEPC-treated H2O • Microcentrifuge
• Vortex
• Pipette
• Pipette tips
 Quality of RNA

• To assess the quality of RNA extraction

electrophoresis is used.

• Crude and refined RNA

• Poor quality RNA will not perform well

in PCR
Concentration measurement
• RNA absorbs UV light with wavelengths of 260 nm due to the resonance
structure of the purine and pyrimidine bases

• The A260/A280 provides insight regarding the type of nucleic acid present
as well as providing a rough indication of purity. Ratios between 1.8 and
2.1 for A260/A280 are accepted as indicating pure RNA.

• Typically, protein contamination can be detected by a reduction of this ratio

and increased ratio can indicate solvent contamination
Concentration measurement
Concentration (ng/ul) = [Absorbance (AU) x Extinction coefficient] / Path length (cm)

A= ε. b.c
BLANK/DILUENT A260/A280RATIO
• A = absorbance at a particular wavelength
DEPC-treated water 1.60
ε = extinction coefficient (pH 5-6)
b = path length of the spectrophotometer Nuclease-free water 1.85
c = concentration of sample (pH 6-7)
TE (pH 8.0) 2.14
 Concentration measurement
Purity is determined by calculating the ratio of absorbance at
260 nm to absorbance at 280 nm.
Purity of the RNA (260/280 nm) =

Nanophotometer

Concentration of RNA sample (μg/ml) =40x A260

Spectrophotometer

Concentration of RNA sample (μg/ml) = 40 x A260 x dilution

factor
Method to write
 References
• Chomczynski P, Sacchi N. Single-step Method of RNA Isolation by Acid Guanidinium Thiocyanate-
Phenol-Chloroform Extraction. 1986. Analytical Biochemistry. 162: 156 -159.

• Ansubel FM, Brent G, Kingston RE, Moore DD, Seidman JG, Smith JA, Stahl K. Guanidine Methods
for Total RNA Preparation. Current Protocols in Molecular Biology Vol. 1.2002. John Wiley and Sons
Inc. Canada. Pg 4.2.1– 4.2.9.

• Bowtell D, Sambrook J. Guanidinium-based Extraction of RNA. DNA Microarrays A molecular

cloning manual. 2003. Cold Spring Harbor Press. New York. Pg 265 – 280.

• RNA: The Other Nucleic Acid. An Educational Kit for the Isolation and Analysis of Total RNA.
Ambion Inc.