Pathway-Centric Tools and Technology™

RNA preparation for successful gene expression profiling

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:

  1. Alberts, B. et al. (1994) Molecular Biology of the Cell, 3rd ed., New York: Garland Publishing, Inc.
  2. 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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