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Why is RNA quality important?
The measurement of diverse mRNA populations in a cell
population of interest is called gene expression profiling. Many popular RNA
profiling techniques such as real time RT-PCR and microarray analysis require
generation of a cDNA copy of the RNA sample through a reverse transcription
reaction. Therefore, the good RNA quality is essential for the success of
these applications because degraded RNA or RNA that contains impurities will
perform poorly in most enzymatic applications. A typical mammalian cell
contains 10-30 pg total RNA. Approximately 360,000 mRNA molecules are present
in a single mammalian cell, with approximately 12,000 different mRNA species
per cell. Some mRNAs comprise as much as 3% of the mRNA pool whereas others
account for less than 0.01%. These low abundance mRNA species may have a copy
number as low as 5-15 molecules per cell. However, these rare species may
account for as much as 11,000 different mRNA species, comprising 45% of the
mRNA population (1).
RNA Integrity
Although mRNA is often the RNA species of interest, the fact that mRNA only
comprises 1-3% of total RNA sample makes assessing the integrity of mRNA
difficult in a total RNA sample. One can use Northern analysis or RT PCR to
study only the integrity of an individual transcript in a RNA sample. Since
the ribosomal RNA (rRNA) makes up more than 80% of total RNA samples, the
integrity of the major rRNA species (18S and 28S for mammalian rRNA) is
routinely used to assess the RNA sample integrity. The most common method of
assessment is to analyze a total RNA sample by denaturing agarose gel
electrophresis. The ethidium bromide-staining pattern of intact total RNA
will have clearly defined 28S and 18S rRNA bands. The ratio of 28S:18S band
intensities should be close to 2. Partially degraded RNA appears as smeared
rRNA bands with increasing lower molecular weight smear. The Agilent 2100
Bioanalyzer is an alternative tool that provides better and more consistent
assessment of total RNA quality. The instrument uses a combination of a
nucleic acid intercalating dye and capillary electrophoresis to analyze both
RNA concentration and integrity simultaneously.
Residue Contaminants
Even with freshly prepared, intact RNA, contaminants in RNA samples sometimes
interferes enzymatic applications. For example, phenol or CsCl2 contaminants
can interfere with the reverse transcription reaction. Some contaminants that
are co-purified with RNA may introduce undesirable side products during
downstream applications. For example, genomic DNA contamination may introduce
background signal in a RT-PCR assay. rRNA constituting more than 80% of total
RNA may compete for reagent in a reverse transcription reaction and generate
unwanted cDNA products.
While a lot of the contaminants like organics, metal ions, lipid,
polysaccharides cannot be easily detected, some contaminants can be detected
using various means. For example, genomic DNA and small RNA species can be
visualized with gel electrophoresis and the Agilent 2100 Bioanalyzer.
Significant protein contamination will result in a lower 260nm/280nm
absorbance ratio (Pure nucleic acid will have 260nm/280nm ratio greater than
1.8). Table 1 lists some of common contaminants and their sources.
| Contaminants |
Contaminant Source |
| Residue organics (e.g. phenol, Alcohol) |
Preparation reagent |
| Metal ions |
Preparation reagent |
| Proteins (e.g. nuclease) |
Original sample |
| Genomic DNA |
Original sample (e.g. nucleic acid rich tissue such as
spleen, thymus and tissue culture cells) |
| Lipid |
Original sample (e.g. neuronal tissue, plant) |
| tRNA and rRNA |
Original sample |
| Polyphenolic acid, lipopolysaccharides |
Original tissue (e.g. Plant) |
| Proteoglycans and polysaccharides |
Original sample (e.g. connecting tissue) |
Table1: common contaminants and their sources
Considerations when choosing a RNA purification method
When purifying RNA sample for gene expression analysis, one has to consider
the source of the sample and the pros/cons of individual purification
methods. Sometimes modifications of the original protocols or using
combination of two purification techniques are needed to obtain high quality
RNA.
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.
Pros, Cons and Remedies
Total RNA preparation methods such as phenol based extraction or silica gel
based chromatography are very easy to carry out and generally have high
yields of total RNA. On the other hand, contaminants mentioned in the last
section will co-purify with total RNA. For example, genomic DNA is a common
contaminant in total RNA preparations using Trizol or RNeasy reagents.
Furthermore, high lipid, nucleic acid, proteoglycan and polysaccharide
content in certain tissues may affect total RNA yield and introduce
inhibitors for subsequent enzymatic applications. Residual contamination of
phenol or alcohol is also detrimental to downstream applications. To minimize
these problems, various modifications have been introduced to improve the
performance of total RNA preparation methods. For example, DNase treatment
during or after RNA preparation is a way to eliminate genomic DNA
contamination. Phase Lock Gel (Eppendorf) can be used to decrease protein
carry over during organic/acqueous phase separation. Centrifugation of crude
homogenates at 12,000Íg at 4oC for 10 minutes help pellet polysaccharides
and DNA; Precipitate the RNA by adding 0.25 ml of isopropanol and 0.25 mL of
0.8 M sodium citrate and 1.2M NaCl per milliliter of TRIzol reagent keeps
proteoglycans and polysaccharides in a soluble form yielding a cleaner
product when tissues rich in these substances, such as liver, are used as the
RNA source (2).
Preparation of enriched poly (A)+ mRNA is an alternative to total RNA
preparation. Since rRNA is the major component of total RNA, it will compete
with mRNA during the cDNA synthesis process. Enriched mRNA samples are also
suitable for microarray gene expression profiling experiments. SABiosciences
recently developed the ArrayGrade mRNA Purification Kit that combines the
oligo-dT affinity purification technique with a magnetic microparticle
technology. The Oligo-dT14 is immobilized on 1 micro, super-paramagentic
microparticles. The oligo-dT microparticles are uniform, colloidally stable
and non-porous spheres that offer high surface area and can remain in
suspension to facilitate the interaction between the poly (A) tail and
oligo-dT. In contrast, the larger and heavier magnetic beads offered by other
commercial vendors have smaller surface areas and will settle too quickly in
solution. SABiosciences' ArrayGrade mRNA Purification Kit is a versatile tool
that can be used to prepare high quality poly (A)+ mRNA with improved yield
from tissues and cultured cell samples. Figure 1 shows experiment results of
focused microarrays using ArrayGrade mRNA Purification Kit and other
manufacturer's total RNA extraction kit. The ArrayGrade mRNA is ideal for
using in gene expression analysis such microarray and real time RT-PCR.
Figure 1: Focused microarray analysis using mRNA isolated with ArrayGrade
mRNA Purification Kit and a total RNA Extraction Kit. Human Cancer
PathwayFinder Gene Arrays (SABiosciences Cat# HS-006) were hybridized under
identical conditions with biotin labeled cDNA probes prepared from 1000 Hela
cells using ArrayGrade mRNA Purification Kit (Panel A) or Trizol (Panel B).
The arrays were detected using Linear Amplification Kit (Cat# L-03) and
Chemiluminescent Detection Kit (Cat# D-01)
Product Information:
| ArrayGrade mRNA Purification Kit |
GA-002 |
| 12-hole Magnetic stands |
GA-003 |
References:
-
Alberts, B. et al. (1994) Molecular Biology of the
Cell, 3rd ed., New York: Garland Publishing, Inc.
-
Xiang. C and Brownstein MJ. (2003) Methods in Molecular
Biology, Vol 224: Functional Genomics: Methods and Protocols. Totowa, NJ:
Humana Press, Inc.
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