Principles And Guidelines

The genome of higher eukaryotes contains close to 50,000 genes, of which between 10% and 15% are believed to be expressed at a given time in a cell to determine the regulatory mechanisms that control cellular processes controlling our lives such as development and differentiation, homeostasis, response to insult, cell cycle regulation, aging, programmed cell death, and pathological changes such as cancer. Monitoring the pattern of gene expression under various physiological and pathological conditions is a critical step in understanding these diverse biological processes, and comprehending the mechanisms involved allows for needed interventions. Because of the large numbers of expressed genes, powerful tools are needed to characterize the overall pattern of gene expression.1-1-1

Older methods of identification of differentially expressed genes relied on differential or subtractive hybridization (SH), which although sensitive requires large amount of RNA, is error prone, nonsystematic, laborious, and time consuming, and results are not seen until the end of the process.[2]

The original DD protocol utilized the idea applied earlier for random amplification of fingerprinted genomic sequences. It was published in 1992 by prominent investigators at the Dana-Farber Cancer Institute in Boston, MA.[3] The principle of the method is to detect different types of gene expression patterns using three techniques: 1) RT of DNase I-treated total RNA (to remove any chromosomal DNA) using anchored primers, 12-mer long consisting of a stretch of 11 Ts plus one last non-T base to anchor primers to the pol (A)+ tail of many RNAs. Use of total RNA is preferable to mRNA as it involves less preparatory steps and avoids background smearing;[2] 2) choosing 5' arbitrary 10 mers that hybridize to cDNA in a degenerate manner for setting lengths of cDNAs corresponding to mRNAs (tags) to be amplified by PCR. For a 5' primer of arbitrary base sequence, annealing position to cDNA should be randomly distributed from the pol (A)+ tail. Therefore the amplification provided from various mRNAs will differ in length; and 3) employing sequencing gels for isolation of cDNAs. The aim is to obtain a tag of a few hundred bases, long enough to uniquely identify mRNA, but short enough to electrophoretically separate by size. Pairs of primers are selected so that by probability each will amplify DNAs from 50 to 100 (average ~ 75) mRNAs, as this number can be adequately displayed on one lane of the gel.[3] This method was later on named DD-PCR[1] (Fig. 1), to distinguish it from other DD methods discussed below.

Problems intrinsic to DD were soon encountered such as high noise level due to smearing, misrepresentation of rare messages, bias toward high copy number mRNAs, not revealing differences due to mutational changes, incomplete cDNAs, contamination of purified PCR fragments by unrelated DNA sequences, and additional bands generated by arbitrary primers alone from palindromic

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5-AAGCI I I I I I I I I I IG-3* (H-T„G) dNTPs y MMLV reverse transcriptase

5'-AAGCTTGATTGCC-3' (H-AP 1) 5'- ® AAGCI I I I I I I I I I IG-3' (RH-T„G) dNTPs ■X Taq DNA polymerase

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5-AAGCI I I I I I I I I I IG-3* (H-T„G) dNTPs y MMLV reverse transcriptase

5'-AAGCTTGATTGCC-3' (H-AP 1) 5'- ® AAGCI I I I I I I I I I IG-3' (RH-T„G) dNTPs ■X Taq DNA polymerase

Negative electrode (-)

Positive electrode (+)

Fig. 1 Schematic of fluorescent DD-PCR method. Total RNA is isolated from cells in culture or tissues. Then, I. A reverse transcription reaction is carried out using one of three specific one-based anchored oligo-dT primers (e.g., H-TnG). II. PCR amplification is performed by using the corresponding fluorescent-labeled anchored oligo-dT primer (RH-TnG; R, rhodamine labeling) in different combination with arbitrary 13-mer primer (H-AP1). III. The amplified PCR products are separated on a denaturing polyacrylamide gel, and the fluorescent-labeled cDNA fragments visualized by a fluorescence scanner. A differentially expressed cDNA fragment in the electropherogram is denoted by an arrow. (Adapted from Ref. [7] with permission.) (View this art in color at www.dekker.com.)

Negative electrode (-)

Positive electrode (+)

Fig. 1 Schematic of fluorescent DD-PCR method. Total RNA is isolated from cells in culture or tissues. Then, I. A reverse transcription reaction is carried out using one of three specific one-based anchored oligo-dT primers (e.g., H-TnG). II. PCR amplification is performed by using the corresponding fluorescent-labeled anchored oligo-dT primer (RH-TnG; R, rhodamine labeling) in different combination with arbitrary 13-mer primer (H-AP1). III. The amplified PCR products are separated on a denaturing polyacrylamide gel, and the fluorescent-labeled cDNA fragments visualized by a fluorescence scanner. A differentially expressed cDNA fragment in the electropherogram is denoted by an arrow. (Adapted from Ref. [7] with permission.) (View this art in color at www.dekker.com.)

sequences within a mRNA molecule; all led to high false-positive rates.[1-7] Therefore many modifications were periodically made to the original protocol to improve these intrinsic problems and to achieve specificity and efficiency as discussed below.

The original primer design was two-base anchored primer, but it resulted in suboptimal amplifications.1-2-1 Three one-base anchored oligo-dT primers differing only at the last 3' non-T base are now used. This modification cuts down the number of RT reactions needed for each

RNA sample and minimized the redundancy and under-representation of certain RNA species due to the degeneracy of the process. Substitution of decamer arbitrary primers with rationally designed 13-mers has increased the accuracy of priming.[5] Furthermore, even longer primers (25-29 mers) and optimal dNTPs concentrations were reported to allow arbitrary priming after an annealing temperature of 40°C in the first PCR cycle, followed by more stringent PCR annealing at 60°C to provide specificity. Additionally, use of hot-start PCR and other thermostable enzyme mixes suitable for long-length PCR resulted in highly reproducible and representative cDNA bands.[6] Moreover, the introduction of a restriction site at the 5' end of both the anchored and arbitrary primers facilitated cloning of the cDNAs.[4]

Initially, isotopes labeled oligos, a-[35S] dATP and later on [32P]-end-labeled oligo-dT primer or a-[32P] dATP, were used to autoradiographically detect amplified PCR products on sequenced gels.[2] Introduction of nonisotopic methods using digoxigenin and fluorescent dyes such as tetramethyl rhodamine allowed coupling of the display with digital data analysis, resulting in increased throughput.[2,7] Use of nondenaturing 6% PAG (i.e., without urea) to reduce double bands into a single band was found to reduce band complexity and eliminated several bands of DNA molecules derived from different fragments that occupied the same position in the gel.[8] Differential display-polymerase chain reaction is only capable of determining the 3' region of the gene, so full-length cDNA can only be achieved either by doing rapid amplification of cDNA ends (5'-RACE) or by probing a cDNA library.[1,7]

Differential display was reported to tolerate a broad range of annealing temperatures and elongation times. However, the major factors that impacted the reproduc-ibility of the method were low concentration of dNTPs and random primers, which made PCR amplifications susceptible to pipetting errors. A final concentration of dNTPs >2 mM and arbitrary primers to 0.2 p.M improved the reproducibility of DD.[9] In a recent study utilizing cervical cancer cell line Caski, it was found that—for most primer combinations—fourfold less cDNA and only 25 high-stringency PCR cycling produced reproducible complex band patterns with intensities that reflected 2- to 10-fold differences in expression levels (the most common levels of regulation).[10] To reduce or exclude the bands that are subject to statistical noise from consideration, it has been suggested to start with enough cDNA (i.e., about 30,000 to 100,000 molecules) to obtain 15 ng/mL of amplified product in 25-27 PCR cycles.[11] Although some publications reveal that as little as 1.1-fold amplification is detectable by DD-PCR, the threshold is not precisely known as an upper detection limit may be reached in this technique.[11]

If we consider that there are 15,000 genes expressed in a cell and each fingerprint contains ~ 70 mRNA species, then in an ideal situation if all mRNAs have the same probability of being displayed in each round, ~ 600 fingerprints (or 45,000 bands) are required to cover 95% of all mRNAs.[8] For a comprehensive analysis of all mRNA species in a given cell, statistical modeling that predicted at least 240 different DD primer combinations is needed.[3] Recently, an empirical determination of the comprehensiveness of DD-PCR in Chinese hamster ovary fibroblast HA-1 cell line that received (or not) hydrogen peroxide treatment used saturation DNA screening of 324 primer combinations. Results showed that a 100% comprehensive analysis by this technique is not possible regardless of the number of primer combinations. This may be due to a selective resistance to the identification of certain sequences by DD.[12]

Once the first few primer combinations have been tested, the results of the display should be examined keeping in mind that a redesign of the experiment should be made when > 5% of the transcripts are differentially expressed, or if no differences exist.[11] It is instructive to keep in mind a few guidelines when performing DD-PCR:

1) drastically different conditions should not be compared;

2) the DD reaction should be repeated for each sample to control for false positives; and 3) multiple samples should be used for each DD experiment in order to provide internal controls.[13]

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