How Do You Know if a Gene Is Expressed in a Gel Box

Reprod Biol Endocrinol. 2004; two: 68.

Methods to find out the expression of activated genes

Sten Z Cekan

¹Karolinska Institute, Department of Adult female and Child Health, Division of Reproductive Endocrinology, Karolinska University Infirmary, Building L5, 17176 Stockholm, Sweden

Received 2004 Jul 8; Accepted 2004 Sep 23.

Abstract

This review deals with the methods of identifying genes that have been activated by inner or outer impulses. The activation and subsequent expression of a gene can be detected by its transcription into a corresponding messenger ribonucleic acid (mRNA). Principles of the methods for identification of individual activated genes, too as groups of activated genes are described, the former methods beingness mostly based on subtractive hybridization and serial analysis of gene expression (SAGE), the latter on microarrays. Examples of gene activation past the hormone 17beta-estradiol (E2) are given.

Introduction

In previous reviews, methods for the measurement of receptors and their interactions with other transcription factors and genes were described [1-iii]. In this review, cistron activation is discussed with a detail accent on the methods enabling detection of the activated, turned-on, genes. The action of the hormone 17beta-estradiol (E2) is taken as an instance of the function of many other minor-molecule compounds in gene activation and in the expression of the activated factor.

The life of humans and animals is influenced by the activity of a series of genes that are kept in a silent state, or are activated, depending on the temporary needs of the body. This switching on and off of each gene is executed past an assembly of transcription factors forming a transcription initiation complex (TIC).

Examples of such transcription factors are estrogen receptors (ER-alpha, ER-beta, and possibly other isomers) that, before being incorporated into a TIC, have to be activated by E2. This hormone itself is synthesized, when an initial signal is given, by virtue of an activation of a series of appropriate genes. Via ER, E2 has manifold biological effects. Biological targets of E2 are, inter alia, blood vessel walls [iv-8], claret platelets [ix], bone [7,x-12], breast cancer cells [13], central nervous organisation [seven,fourteen,15], retinal paint epithelium [sixteen], synthesis of clotting factors [17].

It is axiomatic that E2 is associated with many biological effects and that many genes must be involved. Consequently, ER must be able to bind to DNA segments, called response elements, in the neighborhood of various genes. The response elements participate, together with other transcription factors, in the formation of TICs that are specific for each gene.

An important problem, currently studied in many laboratories, is to find out which genes are activated in various circumstances. The methods that solve this trouble are based on a comparative (differential) approach. A test (target) sample, containing active genes is compared with a control sample in which the genes have not been activated. Using this approach, the active genes are singled out amidst the multitude of inactive genes. However, the comparisons may reveal the reverse of activation, i.due east., downregulation of genes.

More often than not, the activeness of a gene is characterized by its transcription into mRNAs every bit the first pace leading to the synthesis of specific proteins. Non-activated genes in the control tissue do not produce any corresponding mRNAs. In well-nigh methods, the mRNAs prepared from the examination and control tissue are each reverse transcribed into the corresponding complementary deoxyribonucleic acid (cDNA), in order to enable a substantial increase of the material for analysis by polymerase chain reaction (PCR) [two]. As almost methods practise not operate with total mRNA transcripts, merely with shorter sequences, the allotment of such sequences to known (or unknown) genes has to exist constitute by advanced computer programs and factor databases.

The methods used for the identification of active genes are sketched below. Included are fifty-fifty methods that accept not notwithstanding been used for the identification of E2-activated genes. It has to exist mentioned that just principles, not technical details are dealt with in this review. Neither the techniques of cloning or of identification of genes by sequencing are described here. The readers who are not familiar with these techniques are advised to consult appropriate textbooks [e.chiliad., [18]]. The dedicated calculator programs and databases that are needed for the identification of sequences or genes volition not be described hither either. These tin can be institute in the references quoted beneath. It will only be mentioned here that the large databases are GenBank http://www.ncbi.nlm.nih.gov and Celera http://www.celeradiscoverysystem.com.

Activated (expressed) genes can be constitute past comparison of gene contents in the examination and command tissues. There are substantially two approaches for finding activated genes: (i) an individual identification, or (ii) an identification of expression profiles after hybridization to a gear up of known gene fragments (probes) attached to chips in microarrays.

Private identification

This approach means that genes are identified individually, fifty-fifty if several genes can eventually be picked up after cloning. There are several methods that tin can be used.

Differential display

Differential display seems to exist the technically simplest method. Its name stems from the end-point that is a comparison of a side-by-side display of the examination and control preparations by electrophoresis. In its basic class, total RNA of the test and control samples is separately subjected to reverse transcription into cDNA that, in turn, is PCR-amplified using arbitrarily called primers. The products are applied to a gel electrophoresis and the band(s) that are specific for one of the preparations are cut from the gel, further amplified by PCR (using the same primers) and eventually sequenced [nineteen].

In a more advanced version, mRNAs of the test and control cells are separately opposite transcribed to cDNA (Fig. ​ 1). Each transcription is carried out in the presence of a oligo(dT) primers, directed to the poly(A) tail at the 3' terminus of the mRNA and constructed as 5'(NMT11)iii' where Northward can be guanine (Thou), adenine (A), thymine (T), or cytosine (C), and M is G, A, or C [20-22]. The primers with K residues are superior to those having one C remainder. Those ending in A or T are the least efficient. With use of an arbitrary decamer every bit the 2nd primer, a PCR is carried out to amplify the transcript in order to obtain a sufficient working material. This is usually done in the presence of a radioactive nucleotide. Other methods are commonly used, such equally silverish staining. Amplified Dna fragments are separated on a denaturing polyacrylamide gel, the exam training side by side with the command. Each ring differing from those seen in the control electrophoresis is then used for sequencing, subcloning, or as a probe for cDNA library screening. Large amount of results tin be obtained depending on the variation in N and G nucleotides. In spite of the basic simplicity of the procedure, the fourth dimension and workload can be considerable, depending on the number of NM combinations tried.

Principle of a differential display. Test and command mRNA are separately reverse transcribed in the presence of anchored oligo(dT) primers containing nucleotides N and M in various combinations (run across the text). The aforementioned primer and an capricious decamer are and then used as primers in a PCR. The products are subjected to electrophoresis (PAGE). An boosted band (see pointer) in the test sample represents a gene that had non been activated in the control sample. A11 and T11 denote eleven A and T molecules, respectively.

Subtractive hybridization with hydroxylapatite separation

The test mRNA is reverse transcribed into cDNA [23]. This is hybridized with the mRNA of the command sample (Fig. ​ 2). A portion of the exam cDNA (corresponding to the activated gene) does not find whatsoever complementary part in the mRNA of the control sample and remains non-hybridized as a unmarried-stranded cDNA (ss-cDNA). This can be isolated by chromatography on a hydroxylapatite column. The hybridization of the isolated ss-cDNA with control mRNA followed by another chromatography can be repeated to increase the purity of the isolated product [23]. A cDNA library is produced and the subtracted sequence eventually identified. Alternatively, a second hybridization of the isolated ss-cDNA is carried out with the original test mRNA giving rise to a cDNA-mRNA hybrid which, after conversion to double stranded cDNA, is inserted into a vector, a cDNA library is synthetic and several specific cDNA clones are isolated, leading to the identification of several genes [24].

Flow-sheet of subtractive hybridisation with hydroxylapatite separation. Examination mRNA is reverse transcribed into a cDNA. This is hybridized with control mRNA. The non-hybridized portion of the single-stranded sequence of test cDNA is separated by chromatography on hydroxylapatite (HAP) and farther candy.

In another variant [25], the examination and command mRNAs are both reverse transcribed into cDNA. cDNA of the examination sample is hybridized with cDNA of the control sample. The non-hybridized part of the test cDNA is a single-stranded DNA that is separated by hydroxylapatite. The unmarried-stranded Dna is cloned into a vector to produce a subtracted library. Clones with a potent hybridization signal to the subtracted probe are selected and sequenced.

Subtractive suppression hybridization with PCR

Isolation of a single-stranded test cDNA is not needed in this method. mRNAs of the test and control samples are prepared and each is contrary transcribed into cDNA. Each transcript is digested with the enzyme RsaI to obtain shorter, blunt-ended fragments. The examination cDNA is divided into ii portions (see Fig. ​ 3). One of them is ligated with Adapter A, the 2d with adapter B. Each portion is hybridized with an excess of command cDNA. A mixture of hybridization products is formed (Fig. ​ three). A tiny fraction of cDNA remains unhybridized, unmarried-stranded. This is a fragment that may exist called specific, or differentially expressed, or subtracted. It originates from the gene that had been activated. It is absent-minded in the control sample. This specific fragment is spring either to Adapter A or B in the two portions. In the 2d hybridization, the portions are mixed. After annealing, a small corporeality of the specific fragment is obtained double-stranded. It contains Adapter A on the 1 end and Adapter B on the other. After calculation primers specific for the Adapters, the ends are filled and the specific fragment is amplified by PCR to make certain that sufficient amounts are available for a farther processing. Cloning, sequencing and comparing with a gene database establish the identity of the cistron(due south) [26,27] [http://www.clontech.com – "PCR-Select Subtraction kit"]. In contrast to the above methods, the primers for PCR amplification are clearly defined, avoiding thus bug with random primers. This method was used in a number of studies, such as the identification of genes upregulated in rats by E2 and progesterone handling [28]. A predecessor of this technique is the "representational difference assay" [29,xxx].

Outline of subtractive suppression hybridisation with PCR. Exam cDNA and command cDNA are digested with RsaI. The exam cDNA sequences are divided into two halves, one of them being ligated with Adapter A (empty squares), the second i with Adapter B (filled squares). Each half is hybridized with control cDNA. The single-stranded (not-hybridized) sequences of both halves (denoted by asterisks) are annealed in a second hybridization step, primers to the Adapters are added and, after PCR, cloning and cistron identification are carried out.

Expressed sequence tags (EST)

To describe the EST method, the post-obit example is given. cDNA libraries were prepared by reverse transcription from mRNAs of the tissues to be examined [31]. The libraries were converted to plasmids, transfected into Escherichia coli and plated. Hundreds of clones were picked at random. These were subjected to sequencing, followed by estimator matching to known genes listed in the GenBank database. The average length of a sequence was 397 bases; ESTs longer than 150 bases were constitute to be most useful for similarity searches and mapping.

Subtractive hybridization (run across above) was used to isolate the ESTs specific for ane of the libraries. For case, a fibroblast cell line cDNA library was hybridized with a hippocampus library; the common sequences were removed and the specific hippocampus sequences remained. Using the EST method, more than than 2000 human brain genes were identified [32].

Serial Analysis of Gene Expression (SAGE)

The SAGE allows series analysis of factor expression, an analysis of thousands of transcripts. It is based on the assumption that a brusque nucleotide sequence 10 base pairs (bp) – a tag – contains sufficient information to uniquely identify a transcript. In this respect SAGE differs from the EST arroyo.

The principle of SAGE is as follows: mRNA is reverse transcribed into cDNA with use of a biotinylated primer, the cDNA is cleaved with a brake endonuclease and the 3' portions are and so isolated by binding to streptavidin chaplet [33]. In some other version(http://www.invitrogen.com) (Fig. ​ 4), mRNAs are captured prior to opposite transcription on oligo(dT) magnetic beads. Double stranded cDNAs are synthesized and digested with the restriction endonuclease NlaIII that cleaves well-nigh transcripts at to the lowest degree once. The part attached to the magnetic dewdrop is farther processed. The reaction mixture is divided into two portions. The portions are ligated via a brake site R to an adapter A and B, respectively, each consisting of 40 bp. Taking advantage of the brake sites R, both portions are broken with the brake enzyme BsmFI in the altitude of 14 bp. In this way "tags" are formed. Out of these 14 bp, 4 bp are a not-specific segment GTAC. These tags are blunt-ended with the Klenow fragment of DNA polymerase I. The 2 separate pools of tags are ligated together via a blunt-end ligation to produce "ditags". The ditags, flanked by the adapters A and B, are amplified past PCR with use of primers for A and B. The adapters are removed by the enzyme NlaIII and the ditags are concatenated. The resulting concatemers (a series of linked ditags) are cloned into a plasmid vector to create a SAGE library. Individual clones are then sequenced. SAGE is carried out for each sample to be compared.

Period-sail of SAGE. mRNAs are captured on oligo(dT) magnetic beads (open up ovals). Double stranded cDNAs are synthesized. They are digested with Nla Iii. The product is divided into two halves. These are ligated to forty bp adapters AR and BR, respectively. Both adapters contain a sequence R that is a recognition site for the brake enzyme BsmFI. This cuts a 14 bp sequence 3' of the site, forming a 10 bp tag. After cleavage with BsmFI, the tags are ligated to form a production containing a ditag (the points of ligation are denoted by filled circles). This is amplified using primers complementary to A and B. The AR and BR adapters are cut away with Nla Iii to release a ditag. These are ligated to class concatemers containing multiple ditags. The concatemers are cloned and sequenced.

Cheers to the concatenation, many tags can be detected in a single clone [33]. Every bit each tag is supposed to uniquely identify a transcript, SAGE can generate a comprehensive profile of gene expression. Indeed, many unique transcripts were identified with utilize of SAGE tags [34]. The method is particularly useful for detecting genes of low level of expression or in rare tissues (e.k., early embryo) [35,36]. In addition, the amount of individual tags provides quantitative estimates of gene expression [37].

Still, the specificity of detection of genes with utilise of the short tags is non absolute. At that place are ii main problems [38]. The first 1 is that many SAGE tags take no match to known sequences in databases. These tags may correspond so far unidentified genes, but their shortness makes it difficult to characterize the genes. The 2d problem is that the SAGE tags may find multiple matches in the databases [39,40]. Therefore, attempts have been made to increment the specificity by prolongation of the tags by various methods.

One such method is chosen GLGI (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) [34,38,twoscore]. The main feature of this method is the utilise of a SAGE tag as the sense primer for the PCR of a segment of cDNA. An anchored oligo(dT) serves as an antisense primer. In this fashion a cDNA "tag" of up to several hundred bases is created. Still, this method does not seem to ameliorate the specificity of SAGE because even "not-specific" tags are co-amplified.

Better of seems to be another variant of SAGE, the LongSAGE [41]. This is based on the use of tags 21 bp (out of which 4 represent a restriction site), tags longer than those in SAGE. The prolongation of tags is achieved by the use of the restriction endonuclease MmeI. The longer tags increase the power of identification of genes, while not diminishing the sensitivity of SAGE given by the use of PCR and concatenation. Theoretical calculations showed that >99.viii% of the 21 bp tags were expected to occur only once in a genome.

SAGE was used for the investigation of differences in gene expression in diverse health conditions. In the studies of chest tumors [37], global cistron expression profiles in breast carcinoma cells were compared with those in normal mammary epithelial cells. The patterns of factor clusters in normal tissue were distinctly different from those of tumors of dissimilar stage and histological grade. The most dramatic alter occurred at the normal-to-in situ carcinoma transition. This modify tin exist an of import marker for an early diagnosis. In another study, several genes regulated by estrogen or tamoxifen were identified in an estrogen-dependent chest cancer cell line. One of them was studied closer. It appeared to play a significant part in estrogen-promoted cell growth [42].

Gene profiles – microarrays

The DNA microarray analysis is used to identify profiles of expressed genes in a given tissue and time. Thousands of known cDNA sequences or oligonucleotides are imprinted on a solid support, sometimes called a scrap (e.g., a microscope slide or a nylon membrane), using application robots. Typically, individual spots are 100–300 micrometers in size and are spaced about the same distance apart [43]. More than than 30,000 sequences can be fitted on the surface of a chip. These sequences serve equally probes. Alternatively, the probes are synthesized in situ (60-mers) [44]. By hybridization, test (target) sequences (cDNAs or cRNAs) are bound to the cognate probes. The bones approach is the comparing of degree of hybridization in the command and test preparation. There are ii basic techniques for the detection of hybridization. The control and test preparations are placed on a unmarried chip, or, separately, on two chips.

In the single chip technique [18], mRNAs from the control and exam cells/tissues are separately reverse transcribed. During the transcription processes 2 different fluorescent dyes (due east.m., Cy3 – greenish, Cy5 – scarlet) are incorporated into the control and test cDNAs, respectively. The labeled molecules are mixed and hybridized to the cDNA array. There is a competition for each probe on the chip betwixt the control and test mRNAs. The test cDNAs are selectively spring to some probes, the control cDNAs may exist jump to other probes. With utilize of fluorescence scanning it is possible to distinguish the hybrids with control sequences (exhibiting, e.g., green fluorescence) from the hybrids with test sequences (e. m., red) [45]. Alternatively, the dyes may be reversed, and the command and test cDNAs may exist labeled with the carmine and green dye, respectively. The hybrids that ascend when the control and test cDNA occur in equal amounts may show a yellow fluorescence. The blackness spots indicate no hybridization (Fig. ​ 5). Ane of the commercial companies utilizing this arroyo is Agilent http://www.agilent.com/chem.

Model of a microarray. In a single-fleck technique reverse transcription from mRNAs to cDNAs is separately carried out for the test and command cell preparations. During the transcription one of the fluorescent dyes (e.g., Cy3 – light-green and Cy5 – red) are incorporated into the cDNAs of each training. A mixture of these ii preparations is then hybridised to the corresponding cistron-representing sequences on a chip. The activated genes of the control sample showroom green colour, those of the examination sample provide red spots, equally spring cDNAs can exist visualized by xanthous spots, no hybridization remains blackness.

Using a variant of the method [46,47], certain groups of activated genes could be defined equally predictors of the clinical outcome of chest cancer. Up to 5000 genes were tested for up-regulation (red) or downwardly-regulation (dark-green) in upward to 100 patients with diverse degrees of disease progression. Correlations of disease grades with gene expression profiles were established, and a strategy was provided to select patients who would benefit from adjuvant therapy.

In the 2-chip technique, mRNAs of the exam and command tissues/cells is opposite transcribed into a double-stranded cDNA from which a cRNA is prepared. In the grade of the cRNA synthesis biotin molecules are incorporated [48]. The control and test cRNAs are separately hybridized to two identical chips. The binding is detected by staining with a fluorescent dye coupled to streptavidin. Signal intensities are used to calculate the relative cRNA abundance for the genes represented on the assortment. For comparisons of the intensities on both chips avant-garde computer programs accept to be used. A combination of single-chip and two-chip techniques was practical in a report [51] where two chips and two fluorescent dyes were used.

Commercial systems are available from several sources. For case, Affymetrix (GeneChip) [http://world wide web.affymetrix.com] produce chips by a photolithographic method in which thousands of different oligonucleide probes are synthesized in situ on the chip [49]. A meaty technique has been introduced by the Febit company [50]. In a unmarried benchtop instrument called Geniom a light-activated oligonucleotide microarray synthesis takes place, besides as addition of biotin-labeled cRNA sample, hybridization and fluorescence detection after incubation with streptavidin-phycoerythrin [l]. Other systems for microarray production, target grooming, hybridization and result evaluation are offered by Amersham Biosciences http://www.amershambiosciences.com and Clondiag Flake Technologies http://www.clondiac.com.

Every bit a rule, more than one factor is activated, and a spectrum of genes is discovered either occurring sporadically or in clusters [49]. For instance, when a diseased tissue was compared with a good for you ane, an expression profile, a disease fingerprint, was identified [49]. In the case of breast tumors, a molecular portrait of each tumor was obtained [52], or, molecular profiling (a set of gene clusters) provided predictions of responses to adjuvant handling [46,53]. Gene activation in breast cancer cells in the presence of E2 included, apart from the known estrogen-responsive genes, a series of novel genes expressing growth factors and components of the cell cycle, adhesion molecules, enzymes, signaling molecules and transcription factors [48]. Gene expression patterns of breast carcinomas immune to distinguish tumor subclasses [54]. E2 caused up-regulation of 250 genes in vascular endothelial cells that could be prevented by an inhibitor [55]. In an experimental encephalomyelitis a markedly enhanced gene activation by E2 was noted [56].

Sometimes a technically easier macroarray is used, east.1000., on a 96-well plate [57]. Manifestly, the choice of cistron sequences to be used as probes must exist very selective in this case. This arroyo has been adopted by the SuperArray Bioscience Corporation http://world wide web.superarray.com offering selected profiles of genes in the macroarray format for various areas (east.g., cancer, prison cell cycle, cytokine and inflammatory response, etc.).

Quite ofttimes the cistron identification obtained by an array is confirmed by other methods such as Northern absorb assay [58], or existent-time PCR [43,58][http://www.superarray.com]. A negative identification tin can exist achieved past the use of siRNA (small-scale interfering RNA – SuperArray Corp.). siRNAs are brusque RNA duplexes between 15 to 21 nucleotides in length. In one case transfected into cells, a siRNA targets the mRNA containing an identical sequence and degrades it in a catalytic manner. The degraded message is no longer functional in translation (the biosynthesis of poly peptide) and thus in the expression of the respective gene. SuperArray Corp. provides a line of validated populations of siRNAs in the form of SureSilencing siRNA kits.

Conclusions

The methods described to a higher place tin can suit two purposes. The single-gene methods can detect and identify new, previously unknown, genes, whereas microarrays can handle a dandy number of known genes to institute profiles of their expression.

SAGE seems to take advantages over hybridization-based methods for the studies of factor expression, such as differential display and subtractive hybridization. SAGE is superior to the EST approach in providing loftier efficiency in identifying the genes that are expressed at low levels and that represent a majority of genes in the human genome [36].

Microarray techniques unremarkably discover activation of a multitude of genes – a factor profile – that differs from the contour in control tissues/cells and thus – in medicine – may have a diagnostic and/or prognostic value. Withal, the microarray techniques usually crave commercially produced chips too as specialized equipment and advanced, powerful, computing facilities. Thus they are inappreciably affordable for small or medium-size laboratories unless they take substantial financial resource.

A large question at another level remains and so far unanswered: which is the biological "concatenation of commands" in a given tissue and fourth dimension resulting in the activation of genes enabling the biosynthesis of cornerstones for factor activation, such as ligands (e.m., E2), receptors (eastward.g., ER) and other transcription factors, the unabridged machinery leading to gene activation and expression.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC524190/

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