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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 2038-2044
Department of Medicine, University of California, Los Angeles, CA 90066 and Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, CA 90073
Studies of the regulation of gene expression rely upon techniques for the identification and quantitation of mRNA species coding for specific proteins. Several methods have been developed for this purpose, each offering distinct advantages and disadvantages. Rather than provide a comprehensive review of the current mRNA quantitation techniques, it is the intention of this article to discuss three of the most widely used techniques with particular attention to applications and limitations of each. The three techniques that will be discussed are Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction (RT-PCR).3
Northern blot analysis
INTRODUCTION
Introduction
References
). This method involves fractionating RNA species on the basis of size by denaturing gel electrophoresis followed by transfer of the RNA onto a membrane by capillary, vacuum or pressure blotting (Sambrook et al. 1989). The RNA is then permanently bound to the membrane in an apparent noncovalent interaction via exposure to short wave ultraviolet light or by heating at 80°C in a vacuum oven. RNA sequences of interest are detected on the blot by hybridization to a specific labeled probe. Probes for Northern blot detection generally contain full or partial cDNA sequences and may be labeled by enzymatic incorporation of radiolabeled (usually 32P or 33P) nucleotides or with nucleotides conjugated to haptens such as biotin for subsequent chemiluminescent detection. After probe hybridization and washing to remove nonspecific label, the hybridization signal is generally detected by exposing blots to X-ray film or phosphor storage plates, after prior incubation with chemiluminescent substrates if necessary. The resulting band identified by the probe indicates the size of the mRNA, and the intensity of the band corresponds to the relative abundance. Autoradiograph band intensities may be quantitated by densitometry, by direct measurement of hybridized radiolabeled probe via storage phosphor imaging or by scintillation counting of excised bands.
Ribonuclease protection assay
Quantitative RT-PCR
). Hybridization was performed concurrently with probes for apo A-IV and apo A-I, a related mRNA for which expression levels should be similar in control and transgenic animals. Quantitation of the resulting hybridization signals using a phosphorimager revealed a fivefold elevation in apo A-IV mRNA in intestine of transgenic mice when normalized to apo A-I mRNA levels.
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Fig 1.
Northern blot analysis of apolipoprotein (apo) A-IV and apo A-I mRNA levels in intestine of control (Ct) and apo A-IV transgenic (Tg) mice. Total RNA (10 µg) was fractionated by electrophoresis in formaldehyde, transferred to nylon membrane and hybridized to cDNA probes for mouse apo A-IV and apo A-I (Cohen et al. 1997 ). Hybridization signals were detected by phosphorimaging and were quantitated using ImageQuant software (Molecular Dynamics).
-actin, 
-tubulin, 2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and hypoxanthine-guanine phosphoribosyl transferase. Probes specific for 18S or 28S ribosomal RNA (rRNA) may also be used. The choice of an appropriate standard must be tailored to the specific experiment, as even the expression of ubiquitous transcripts and housekeeping genes may be altered in various experimental treatments and physiological conditions (Finnegan et al. 1993), at different stages in the cell cycle (Mansur et al. 1993), or among different cell and tissue types (Biragyn et al. 1994). Multiple studies have concluded that hybridization with 18S or 28S rRNA probes is a reliable control for sample normalization on Northern blots (Biragyn et al. 1994, de Leeuw et al. 1989, Finnegan et al. 1993). An alternative strategy to hybridization involves densitometric quantitation of ethidium bromide-stained rRNA after transfer to the membrane (Correa-Rotter et al. 1992). This technique eliminates the issue of variability in expression of housekeeping genes, as well as the need for a second round of hybridization. However, it is unlikely to find widespread use as it requires computer-assisted video densitometry of photographic negatives which have been made from the stained membrane.
). With the use of a vacuum filtration manifold, samples are deposited onto a filter in a fixed array of dots or slots. After hybridization and detection by autoradiography, signals may be quantitated by densitometry, phosphorimaging or scintillation counting of filter spots. The relative ease of sample preparation and application permits the analysis of serial dilutions of each sample to allow comparison of the relative mRNA abundance between samples. Although this technique offers the advantage of being simpler to perform than a standard Northern blot, there is a compromise in the stringency with which the resulting data can be interpreted. Without size fractionation of the RNA, the results are blind with regard to the amount of hybridization signal produced by interaction with the target mRNA vs. hybridization to related sequences (such as members of a gene family) and nonspecific hybridization. Thus it is imperative to demonstrate via Northern blot that the probe used for dot/slot blot analyses is specific for the target mRNA without cross-hybridization or nonspecific hybridization to other sequences.
, Sambrook et al. 1989) (see Fig. 2, upper panel). After incubation for several hours, unhybridized probe and sample RNA are enzymatically degraded and the remaining hybrids are electrophoresed through a denaturing polyacrylamide gel and visualized by autoradiography or phosphorimaging. Alternatively, the RNase-resistant hybrids may be precipitated and bound to filters for direct quantitation by scintillation counting (Melton et al. 1984). Furthermore, by performing titration reactions with unlabeled RNA transcripts corresponding to the mRNA sense strand, absolute RNA levels can be determined.
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Fig 2.
Ribonuclease protection assay (RPA). (upper panel) The RPA is performed by hybridization in solution of labeled RNA probe to an RNA sample containing the target RNA. After hybridization, excess single-stranded probe and nontarget RNA is digested by treatment with an RNase mixture. The protected probe-target RNA hybrids may be analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) or by precipitation and filter binding for quantitation by scintillation counting. (lower panel) Structure of a typical transcription template for synthesizing an RNA probe. cDNA sequence corresponding to the target mRNA is subcloned downstream of a bacteriophage RNA polymerase promoter (i.e., T3, T7 or SP6 phage promoters). The cDNA fragment must be cloned in the antisense orientation such that transcription from the phage promoter generates an RNA probe that is complementary to the target mRNA. To prepare the probe, the plasmid is digested at a restriction site within the plasmid multiple cloning site (MCS) to produce transcripts of a defined length (in this case, 250 nt). Hybridization of the RNA probe to the target RNA followed by RNase digestion will produce a 200-nt protected fragment that is distinguishable in size from undigested probe.
). The sensitivity and specificity of the RPA can be attributed to the use of single-stranded RNA antisense probes which hybridize to a defined region of the target mRNA and are labeled to high specific activity (109 cpm/µg). Single-stranded RNA probes are prepared using plasmid vectors containing specific sequences that serve as promoters for bacteriophage RNA polymerases, such as those derived from SP6, T3 and T7 phages (Lee and Costlow 1987). A number of such plasmids are commercially available, most of which contain two unique phage-specific promoters, one on either side of the polylinker sequences. A DNA insert corresponding to the mRNA of interest is cloned into the polylinker and RNA transcripts equivalent to either the sense or anti-sense strand are produced by choosing the appropriate phage RNA polymerase to initiate transcription (Lee and Costlow 1987). Radiolabeled antisense probes are produced by including a radiolabeled ribonucleotide (i.e., 32P-UTP) in the transcription reaction; a corresponding unlabeled sense RNA to be used as a titration standard may be transcribed from the opposite promoter by providing only cold ribonucleotides in the reaction. The size of the RNA transcript is controlled by predigesting the plasmid template with a restriction enzyme that cuts at a single site downstream from the promoter, thus serving to halt transcription due to polymerase runoff.
). RNase-resistant hybrids derived from both probes can be quantitated on the same filter by choosing the appropriate parameters for scintillation counting.
). For absolute RNA quantitation the RPA/filter precipitation method is superior to the related DNA solution hybridization/S1 nuclease method because of its lower background, fewer enzyme artifacts and its use of RNA rather than single-stranded DNA as a standard, thus eliminating problems due to differences in RNA/DNA vs. RNA/RNA hybridization kinetics (Lee and Costlow 1987, Melton et al. 1984, Sambrook et al. 1989).
). Typically, very small amounts of total RNA (1 µg or less) are used for reverse transcription, and a fraction (1/20 to 1/50) of the resulting cDNA is used in the PCR.
, Volkenandt et al. 1992). Because of the sensitivity of PCR, very small amounts of genomic DNA contamination in an RNA preparation may serve as template for amplification and produce misleading results. To avoid this problem, PCR primer pairs should be designed such that the sequence for each primer occurs in a separate exon of the gene of interest; this allows the distinction on the basis of size between products resulting from amplification of cDNA from those which may have arisen from contaminating genomic DNA. It has also been recommended that RNA preparations be treated with DNase prior to cDNA synthesis, and that control reactions be performed in which reverse transcriptase is omitted (Dirnhofer et al. 1995). Perhaps the most important factor that complicates the use of PCR for quantitation is the fact that the true exponential phase of the amplification reaction is of limited duration. As amplification products accumulate, the exponential phase eventually enters a saturation phase where products may approach similar levels irrespective of initial template concentration. Thus, for results to be meaningful, quantitative comparisons must be made during the exponential phase. Following is a discussion of various strategies for using RT-PCR to determine relative and absolute mRNA levels.
Progressive analysis during the exponential phase 
real-time PCR.
One strategy to ensure that PCR products are analyzed within the log phase of the amplification reaction is to examine products at progressive cycles during the reaction. This may be accomplished by manual removal of reaction products (Jaegle et al. 1996, Reue et al. 1997) or by the use of automated systems that permit real-time quantitative RT-PCR (Blok et al. 1997, Gibson et al. 1996, Heid et al. 1996). In its simplest implementation, aliquots are removed from the PCR every couple of cycles beginning at a point where product is undetectable (typically about cycle 20) and extending through the entire exponential phase. Products are then resolved electrophoretically and quantitated by densitometry, fluorescence or phosphorimaging.
Quantitative RT-PCR with endogenous standards.
Two general types of standards have been employed for absolute quantitation of target mRNAs by RT-PCR (reviewed in Foley et al. 1993 Quantitative RT-PCR with exogenous standards.
The second quantitative RT-PCR strategy involves coamplification of an exogenous RNA standard which has identical primer binding sequences as those in the target mRNA template (Becker-André and Hahlbrock 1989 RNA/DNA quantitative PCR.
An alternative to the RT-PCR techniques with coamplification of either endogenous or exogenous RNA standards has recently been proposed (Zenilman et al. 1995 Choice of mRNA quantitation method
Manuscript received 1 June 1998. Initial reviews completed . Revision accepted 3 August 1998.
). As a control, cDNA samples from adipose tissue, monocytes and THP-1 cells were diluted 30-fold and amplified with -actin primers (Fig. 3).
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Fig 3.
Semi-quantitative RT-PCR with progressive analysis of products during amplification. Hormone-sensitive lipase (HSL) mRNA is an abundant mRNA in adipose tissue but a rare mRNA in monocytes. To determine the relative abundance of HSL mRNA in the two tissues, RT-PCR was performed using different amounts of input cDNA, and the appearance of product was monitored at progressive cycles during the amplification. As shown in the upper panel, cDNA samples from adipose tissue, the THP-1 monocyte cell line and monocytes were diluted 1:30 for amplification with control -actin primers, and PCR products analyzed after 28, 32, 36, and 40 cycles. In the lower panel, adipose tissue cDNA was diluted 1:40, and THP-1 and monocyte cDNAs were not diluted. Appearance of product from the undiluted monocyte samples occurred at a similar PCR cycle as for adipose cDNA which had been diluted 40-fold, indicating that HSL mRNA is expressed in monocytes at approximately the levels in adipose tissue. From Reue et al. 1997, reproduced with permission.
). The assay is based on the use of a fluorogenic probe that has both a reporter and quencher dye attached (Livak et al. 1995). The presence of the two dyes together leads to quenching of the reporter dye. However, when the probe anneals to the target cDNA sequence during PCR, the 5` nuclease activity of Taq DNA polymerase cleaves the reporter dye from the probe and a fluorescent signal is emitted. The resulting signal is automatically monitored at every cycle by a system that integrates the thermal cycler, reaction tubes with transparent lids, a laser for inducing fluorescence, and a detection and recording device. For absolute quantitation of target mRNA, an internal control template and corresponding control probe with a unique reporter fluorescent dye is included in each reaction tube (Gibson et al. 1996) (see below for further discussion of internal controls for PCR). Real-time quantitative PCR is extremely accurate, allows high throughput and is less labor-intensive than other methods available. However, at this point in time the cost of the integrated PCR/detection system is prohibitive for most labs which are performing quantitative PCR on a small scale. Thus, the other strategies for absolute quantitation of mRNA by PCR described below are still in most widespread use.
, Raeymaekers 1995, Volkenandt et al. 1992). One involves the coamplification of an endogenously expressed standard RNA that is present in the same mixture as the target mRNA, much like the common practice of using housekeeping genes as normalization standards in Northern blot or RPA analyses. Experimental samples are normalized with respect to the endogenous RNA standard to allow comparison of mRNA levels. The endogenous mRNA standard has the advantage of serving as a control for the extent of RNA recovery and integrity, as well as for sample-to-sample variations in reverse transcription and PCR. As with the other mRNA quantitation techniques, the choice of a standard mRNA depends on the specific application and requires a demonstration that the standard RNA level is invariant under the conditions of the experiment.
).
), of -, -, and 
-actin isoforms in the aorta (Raeymaekers 1995), and of multidrug resistance mRNAs MDR-1 and MDR-2 in multiple myeloma patients (Raeymaekers 1995). These kinds of applications are perhaps the most amenable to internal standard RT-PCR.
, Gilliland et al. 1990, Wang et al. 1989). Termed competitive RT-PCR, this method requires that several reactions be performed with a set amount of sample RNA and increasing known amounts of the standard. The competitor RNA standard is generally prepared by in vitro transcription and its concentration carefully determined. Since both the target and standard must compete for the same amplification primers, the amount of input target mRNA can be determined based on the standard concentration at which amplification products for target and standard are equivalent. Thus, unlike coamplification with an endogenous standard, absolute quantitation is possible. In addition, the use of a single primer pair eliminates many of the problems outlined above for endogenous standards. In particular, initial concentrations of standard and target RNA need not be similar and can differ up to two orders of magnitude (Bouaboula et al. 1992). Several investigators have assumed that the use of the same primer pair for target and standard confers identical amplification efficiency and a constant ratio between target and competitor even beyond the exponential phase (Becker-André and Hahlbrock 1989, Gilliland et al. 1990, Siebert and Larrick 1992). However, other investigators caution against this assumption based on observations that the exogenous standard may have an "amplification advantage" over the target (Volkenandt et al. 1992) and that standard and target products accumulate with different efficiencies (Raeymaekers 1995, Wang et al. 1989, Weisner et al. 1993). It appears that the feasibility of using this technique beyond the exponential phase must be evaluated for each specific standard and target pair.
, Dirnhofer et al. 1995, Raeymaekers 1993 and 1995, Volkenandt et al. 1992, Zenilman et al. 1995). Most agree that in order to serve as a competitive standard RNA, the template must have identical primer sequences as the cellular target mRNA and similar, yet distinguishable, amplicon size. A common strategy for designing competitor templates is to insert or delete a restriction enzyme recognition site within the internal sequences of the template so that the products of the target and standard amplification can be easily resolved by electrophoresis after restriction digestion (Raeymaekers 1995). Although a general recommendation has been to keep the target and competitor amplicons a similar size to avoid differences in amplification efficiency, it appears that in some cases this may not be critical (Bouaboula et al. 1992). A solution to the laborious task of constructing a new standard template for each individual mRNA to be analyzed is to produce a single construct containing multiple PCR primer sequences (Bouaboula et al. 1992, Wang et al. 1989). Specifically, 5' primers for a dozen or so target mRNAs are connected in tandem followed by the complementary sequences of the corresponding 3' primers in the same order. The 5' and 3' primers may be separated by a linker region containing restriction enzyme recognition sequences, and the entire array is cloned downstream of an RNA polymerase promoter to allow production of RNA transcripts in vitro. This type of standard template has been employed to quantitate absolute levels of rare lymphokine mRNAs (Bouaboula et al. 1992, Wang et al. 1989).
, Raeymaekers 1993). This can be achieved by performing a series of reactions in which the amount of input target cDNA is held constant and the amount of standard competitor cDNA is increased over 2-3 orders of magnitude. By including a radiolabeled dNTP in these reactions, target and competitor PCR products can be quantitated by resolving on gels, isolating the fragments and scintillation counting. The PCR product ratio (standard/target) is then plotted on a log-log scale as a function of the input standard amount. This should produce a linear relationship with a slope equal to 1, which serves as a practical indication (but not proof) that both templates are amplified with the same efficiency (Bouaboula et al. 1992, Raeymaekers 1995).
). Discrepant amplification efficiencies between the standard and target are most likely to show up when the amplification reaches the saturation phase and the PCR components decrease below levels required for optimal amplification. Thus, if an invalid titration curve is obtained (that is nonlinear or with a slope other than 1), a possible practical solution is to reduce the number of PCR cycles and/or the initial amount of template (Raeymaekers 1995). A unexpected source of error in generating titration curves is the use of agarose gels for quantitation of radiolabeled ethidium bromide-stained PCR products. A comparison of polyacrylamide and agarose gels for analyzing competitive PCR products has indicated that for the same samples which produce a titration curve with a slope of 1 when analyzed on polyacrylamide gels, analysis on agarose produces a slope of significantly <1 (Bouaboula et al. 1992). This has been attributed to a 20-fold higher background in agarose compared to acrylamide (1000-2000 cpm vs. 50-90 cpm). One reliable approach appears to be quantitation of radiolabeled PCR products on dried polyacrylamide gels with phosphorimager scanning (Raeymaekers 1995).
). As this technique can be utilized for quantitation of either DNA or RNA, it has been named RNA/DNA quantitative PCR (RD-PCR). This technique utilizes genomic DNA present in total cellular nucleic acid preparations as an internal standard against which the concentration of the target mRNA is measured. This allows the determination of the number of mRNA copies per cell at a sensitivity 100-fold greater than for RPA (Zenilman et al. 1995). This is accomplished by carrying out the reverse transcription step using a hybrid primer which contains sequence complementary to the target mRNA at its 3' end and sequence complementary to an intron of the corresponding gene at its 5' end. This results in the production of a cDNA that is tagged for intron sequence; genomic DNA is not affected in the reverse transcriptase step. Competitive PCR is then performed in the presence of the intron-tagged cDNA and genomic DNA using the intron primer in combination with a 5' primer derived from upstream exon sequence to simultaneously amplify the cDNA and genomic DNA. Conveniently, the amplicons generated from cDNA and genomic DNA will be of different sizes. As with RPA, a titration curve can be constructed with in vitro synthesized sense RNA corresponding to the target mRNA.
View this table:
Table 1.
Comparison of mRNA Quantitation Techniques
1
Supported by the U.S. Public Health Service (HL-28481) and the American Heart Association (Established Investigator Award).
FOOTNOTES
2
The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby marked "advertisement"
in accordance with 18 USC section 1734 solely to indicate this fact.
3
Abbreviations used: apo, apolipoprotein; cRNA, unlabeled sense RNA; nt, nucleotides; RD-PCR, RNA/DNA quantitative PCR; RPA, ribonuclease protection assay; rRNA, ribosomal RNA; RT-PCR, reverse transcriptase-coupled PCR.
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LITERATURE CITED |
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Introduction
References
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-actin retropseudogenes interfere with RT-PCR.
Trends Genet.
1995;
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