FR2816322A1 - Separating differentially expressed nucleic acids, useful e.g. for identifying cancer genes, by isolating RNA of one cell that does not hybridize to cDNA from another - Google Patents

Abstract

Separating nucleic acids (NA) that are differentially expressed between a target cell (TC) and reference cell (RC). RNA from RC is reverse transcribed to form RNA-cDNA heteroduplexes, from which the RNA is removed. RNA from TC (amount equivalent to the RNA of RC) is then added to the remaining cDNA to form heteroduplexes and any TC-derived RNA that does not form a heteroduplex is isolated as differentially expressed NA. Independent claims are also included for the following: (a) method for identifying NA sequence, or fragments, differentially expressed between TC and RC by separating and amplifying cDNA, derived from the separated RNA, sequencing, then optionally screening databases for the sequences obtained, or their homologs or complements; (b) method for identifying a protein, or fragment, that is differentially expressed by deducing the sequence from the NA sequences; (c) method for identifying the gene that encodes a protein of (b), from the sequence deduced in (a); and (d) method for quantifying a transcription product, or fragment, of the genes of (c) by identifying as in (a), designing primers and/or probes, then using these for quantification.

Description

<Desc / Clms Page number 1>

METHOD FOR SEPARATING DIFFERENTLY EXPRESSED NUCLEIC ACID BETWEEN A TARGET CELL AND A REFERENCE CELL
The subject of the present invention is a method of separating nucleic acid differently expressed between a target cell, in particular of a tumor nature, and a reference cell. The present invention also relates to a method of identifying said differently expressed nucleic acids as well as a method of quantifying said nucleic acids. The invention finally comprises a method for identifying a gene capable of being involved in the tumor process of mammalian cells, in particular human cells.

 Cellular physiology, such as the regulatory cycles, differentiation or development of a cell, is dependent on the genetic heritage of the cell and the expression of its genes. Thus any change in gene expression for a cell can lead directly to the modification of its physiology and give it a character responsible in particular for regulation, differentiation or abnormal development of this cell. Thus, the identification and characterization of differently expressed gene (s) between a cell having an abnormal character and a cell not exhibiting this abnormal character would undoubtedly provide decisive elements for the understanding of these deregulation mechanisms. , or differentiation or abnormal development of a cell, in particular cancerization processes, and would accelerate the development and marketing of effective compounds against pathologies induced by these differently expressed genes.

Among the methods making it possible to demonstrate a difference in expression of one or more genes between two cells, there may be mentioned, for example, the DSD Method of Differential Substraction Display Method for the isolation of CDNA of genes differently expressed between two cells (Pardinas et al., Anal. Biochem., 257,161-168, 1998).

In this method, described here briefly, the single-stranded cDNA obtained from a sample of purified total mRNAs from target or reference cells is amplified by combinatorial PCR using primers of arbitrary sequence and in the presence of labeled nucleotides. , then, after denaturation, separated by size by electrophoresis on polyacrylamide gel and autoradiographed. After comparison of

<Desc / Clms Page number 2>

 gels obtained for each of the cell samples tested, the cDNAs contained in a band (-) of gel corresponding to RNAs not or under-expressed, compared to the equivalent band of the other sample, are taken, re-amplified, labeled and used as a template for the subtractive hybridization step of the cDNAs taken from the equivalent (+) band of the other sample and re-amplified. The non-subtracted cDNAs, thus corresponding to RNAs only or overexpressed by one of the samples are separated, again re-amplified by combinatorial PCR then cloned and / or characterized.

 If this subtractive method makes it possible to eliminate a non-negligible part of the genes whose expression is redundant in the samples compared, the amplification and re-amplification steps by combinatorial PCR prior to the subtractive hybridization step are tedious and tend to favor the amplification of the cDNAs initially present in large quantities, generally redundant, in the samples tested, to the detriment of the cDNAs corresponding to RNAs which are poorly expressed and rare and which may be the source of the abnormal nature of the sample studied (cf. also Wan et al., Methods in Molecular Biology, Vol. 85, Chapt. 5, pp 45-68, 1997, Humana Press Inc., Edited by Liang, P. and Pardee, AB). On the other hand, the amplification and re-amplification steps by combinatorial PCR, prior to the subtractive hybridization step, generally bring about the generation of cDNA fragments of false-positive type.

 Among the methods making it possible to demonstrate a difference in gene expression, there may also be mentioned the American patent document published under the number US 5,882,874 (Fisher; Paul. B.) which describes an RDSD method of analysis by subtraction Reciprocal Differential Substraction Display method, for the isolation of cDNA from genes expressed differently between two cells from two phage cDNA libraries (Lambda ZAP vector, Stratagene) representative of the RNAs expressed by these two cells.

In this method, the two phage cDNA libraries are mutually subtracted and are then transformed into two bacterial plasmid banks containing the subtracted cDNAs (excised in vivo according to the protocol of the supplier Stratagene). These plasmid cDNAs, after purification, are amplified by combinatorial PCR and the PCR products obtained are then separated by gel electrophoresis. After comparison of the bands obtained for and between each of the two

<Desc / Clms Page number 3>

 samples, the cDNAs corresponding to different bands are taken from the gel, then analyzed and cloned.

 The method described in this patent document most certainly includes a step of cloning the cDNAs in a phage vector which will then be propagated to obtain a stable cDNA library, then, after reciprocal subtraction of these libraries, a new step of cloning the inserts of CDNAs excised into a plasmid vector which will also be propagated to obtain the subtracted cDNA library.

 Such a method, even if it does not include a combinatorial PCR step before the subtractive step, nevertheless includes a prior cloning and amplification (propagation) step which does not make it possible to ensure with a minimum of accuracy of the quantity and the integrity of the cDNA from the two phage libraries which will be used during the subtractive stage, determining parameters for carrying out a relevant subtractive stage whatever the nature and the quantity of the RNAs differently expressed that one wants to separate and characterize.

 Thus, there is today a great need for a reliable and efficient subtractive method of separation and identification of nucleic acids differently expressed between a target cell and a reference cell which makes it possible to avoid any preliminary step d amplification by PCR or by in vivo propagation of the cDNAs corresponding to the RNAs extracted from the sample before the actual subtractive step.

 It is desirable that such a new method also eliminates any step of prior manipulation of RNA / cDNA such as by hydrolysis in the presence of restriction endonucleases followed by artificial introduction (by ligation) of adapters compatible with the cohesive end or not. cohesive enzymatic cleavage sites. Indeed, such types of adapter can introduce small known oligonucleotide sequences which are generally used to hybridize primers for the PCR amplification steps. The use of these types of adapters characterizes many kits on the market (such as the AFLP Amplification Fragment Lenght Polymorphism kit marketed by the company Applied Biosystems or the SMART kits marketed by the company CLONTECH).

The use of these restriction enzymes is based on the assumption that the mRNAs all contain the respective enzymatic cleavage site However, if this

<Desc / Clms Page number 4>

 hypothesis is only correct at 90%, 80%, 70% or less, such a prior manipulation of RNA / cDNA would introduce an analytical pre-selection bias which would exclude a non-negligible fraction of RNA from all of the cellular RNAs, and thus a method comprising such prior manipulations of RNA / cDNA would not reflect the reality of all of the RNAs expressed.

 Such a method should make it possible to lead to the identification of a gene implicated in pathologies associated with the abnormal expression of a gene, in particular cancers, and thus to identify potential therapeutic targets for the search for new drugs effective for the prevention and / or treatment of these pathologies.

 This is precisely the object of the present invention.

The present invention thus relates to a method of separating nucleic acid differently expressed between a target cell and a reference cell, characterized in that said method comprises the following steps: a) the selection of a first target sample and a second reference sample, which samples respectively contain a repertoire of RNA originating from said target cell and from said reference cell; b) the formation of heteroduplex of RNA-cDNA type (of reference mRNA-reference cDNA) from a given quantity of RNA contained in said reference sample by polymerization using a reverse transcriptase; c) removing the RNA from said RNA-cDNA heteroduplex formed in step b); d) the formation of heteroduplex of RNA-cDNA type (of target mRNA-reference cDNA) from the cDNA obtained in step c) by adding an amount of RNA from the selected target sample to step a) equivalent to the given quantity of RNA of the reference sample used in step b); e) separation of the RNA added in step d) non-hybridized heteroduplex type
CRNA-cDNA obtained in step d); the non-hybridized RNAs isolated in step e) containing the RNAs expressed differently between said target cell and said reference cell.

 In step b) of the present method of the invention, the formation of heteroduplex of the RNA-cDNA type from a given quantity of reference RNA will be

<Desc / Clms Page number 5>

 preferably carried out on an aliquot of known volume and concentration (or known quantity) of RNA, taken from said reference sample.

 The term “nucleic acid differently expressed between a target cell and a reference cell” is intended to denote a nucleic acid of RNA type, preferably mRNA, the amount of which is expressed by one of said cells is significantly different from the amount expressed by the other. of said cells, this quantity being able to be greater or less.

 The term “nucleic acid differently expressed between a target cell and a reference cell” is also intended to denote a nucleic acid of RNA type, the expression of which is demonstrated only for one of said cells, in particular an RNA expressed by one of said said cells whose nucleic sequence does not allow its hybridization in the form of heteroduplex with the cDNAs corresponding to the RNAs of the other of said cells. Said RNA expressed by one of said non-hybridized cells in the form of a heteroduplex may correspond to an RNA whose gene is not expressed in the other of said cells or whose gene comprises in its sequence a variation with respect to the gene corresponding in the other of said cells, said variation being sufficient to prevent hybridization.

 By target sample or reference sample, we mean any sample from a biological sample likely to contain nucleic acids of RNA type of animal or vegetable origin, or from microorganism (parasite, bacteria, yeast, fungus or virus), preferably capable of containing RNAs from eukaryotic cells, in particular from mammals such as humans. We will particularly prefer samples from biological fluid (whole blood, amniotic fluid, bone marrow, ...), biological tissue or biopsy, or cells, in particular eukaryotes, taken directly from the organism from which they are derived or obtained from in vitro culture of said tissues or cells (cell line for example).

 Preferably, said target sample and reference sample will come from a fraction enriched in RNA from these biological samples, preferably from a fraction containing purified RNA, preferably messenger RNA, in a sufficient concentration. to allow quantification with precision, preferably with an accuracy of at least 5%, 2.5%, 1% or 0.5%, the amount of RNA contained

<Desc / Clms Page number 6>

 in said samples in step a) and added to step d) of the method according to the present invention, preferably at least 2%, ie 20 ng for a sample containing 1 ug of RNA.

Among the methods making it possible to purify the RNAs, in particular the messenger RNAs (or mRNAs) at a sufficient rate or at a sufficient concentration, from a biological sample for the implementation of the method according to the invention, mention may be made of: but not limited to, the methods of extraction of total RNAs followed by a method of purification by affinity chromatography on an oligo (dT) -cellulose column (or oligo (dU) -sephadex &commat;) for the mRNAs of mammals or followed by a purification method by centrifugation on a cesium chloride gradient, such as for example those described in the document Sambrook et al. (Molecular cloning, A Laboratory Manual, Vol. 1, pages 7.3 to 7.29, Second Edition, Cold Spring Harbor Laboratory Press,
1989).

 In a common manner, use is made of mRNA separation kits such as those sold by the company Qiagen (oligo (dT) beads).

 Among the methods for quantifying with sufficient precision the concentration of RNA present in an aliquot of a preparation of purified RNA sample, mention may be made, but is not limited to, the method of quantification of RNA based on the reading of the optical density at 260 nm as described for example on page 7.17 of the document Sambrook et al. cited above.

 However, for greater precision, it is preferable to use the quantification of RNAs using TaqMan (R) technology (quantitative real-time PCR). This technology makes it possible to measure with an accuracy of the order of at least 1 ng on the one hand the total RNAs (consisting of ribosomal RNAs, messenger RNAs and transport RNAs) and on the other hand the mRNAs . The difference between the measured value (for a given gene such as p2 microglobulin) between total RNA and mRNA testifies to the enrichment in mRNA at the expense of ribosomal RNAs.

 The methods of extraction and purification of RNA from a biological sample, as well as the methods of quantification of RNA are known to those skilled in the art who will be able to choose from these methods of extraction and purification those which are best suited to the type or nature of the biological sample from which the target and reference samples will be taken, and choose from these methods

<Desc / Clms Page number 7>

 quantification of RNA that which will make it possible to obtain the desired precision for the implementation of the method according to the invention.

 For the implementation of the method according to the invention, it will preferably be necessary to control the quality, such as the integrity, of the RNAs thus extracted and purified. Usually, after preparation of the total or messenger RNAs, the quantitative yield of these preparations is obtained by spectrophotometry while the optical absorbance ratio at 260 and 280 nanometers makes it possible to judge the quality of these preparations. The usual methods used to control the integrity of the RNAs obtained may be based on a separation of these RNAs by electrophoresis on agarose gel and on visual inspection of the bands corresponding to the 28S and 18S ribosomal RNAs.

 Mention may also be made of the technique making it possible to control the integrity of the mRNAs based on a separation by gel electrophoresis as described on pages 8.3 and 8.4 of the document Sambrook et al. (Molecular Cloning, a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press 1989).

d'ARN (en volume) prélevée et/ou additionnée. L'homme de l'art saura choisir parmi ces méthodes de quantification d'ARN et parmi les systèmes de manipulation de liquide ceux permettant d'obtenir la précision, la fiabilité et la reproductibilité voulues, à partir des notices techniques fournies par les fabricants ou à partir de tests d'étalonnage, de courbes comparatives et/ou de calculs statistiques qu'il effectuera si nécessaire. By adding an amount of RNA equivalent to a given amount of RNA is meant denoting the addition of an amount equal to a given amount of RNA with the uncertainties related to the accuracy of the determined RNA concentration by the quantification method chosen, and those linked to the precision on the quantity

of RNA (by volume) taken and / or added. Those skilled in the art will be able to choose from these methods of RNA quantification and from liquid handling systems those making it possible to obtain the desired precision, reliability and reproducibility, from the technical manuals provided by the manufacturers or from calibration tests, comparative curves and / or statistical calculations which it will carry out if necessary.

 The invention preferably relates to a method for separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said non-hybridized RNAs isolated in step d) contain RNAs overexpressed or RNAs whose expression is only detectable (or demonstrated) in the target cell compared to the reference cell (gene only expressed in the target sample or expressed in a different form such as a sequence with a truncated fragment or additional, or with a large number of point mutations).

<Desc / Clms Page number 8>

 In a particularly preferred embodiment and in order to obtain both the over-expressed and under-expressed mRNAs in a target cell relative to a reference cell, the invention relates to a method for separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said method comprises the following steps: a) the selection of a first target sample and a second reference sample, which samples respectively contain a repertoire of Target mRNA and reference mRNA from said target cell and said reference cell; b) the formation of heteroduplex of the reference mRNA-reference cDNA from a given quantity of reference mRNA contained in said reference sample and the formation of heteroduplex of the target mRNA-target cDNA from a quantity target mRNA data contained in said target sample by polymerization using reverse transcriptase; c) removing the mRNA from said mRNA-cDNA heteroduplex formed in step b) for each of the samples obtained in step b); d) the formation of heteroduplex of mRNA-cDNA type from the cDNA contained in the sample for each of said samples obtained in step c) by addition: - in the sample obtained in step c) containing the cDNA corresponding to the mRNA of the target sample, of an amount of mRNA of the reference sample selected in step a) equivalent to the given amount of mRNA of the target sample used in l step b), and - in the sample obtained in step c) containing the cDNA corresponding to the mRNA of the reference sample, of an amount of mRNA of the target sample selected in the step a) equivalent to the given quantity of mRNA of the reference sample used in step b); e) the separation for each of the samples obtained in step d) of the mRNA added in step d) not hybridized from the heteroduplexes of mRNA-cDNA type obtained in step d); the non-hybridized mRNAs isolated in each of the samples in step e) containing the differently expressed (over-expressed and under-expressed) mRNAs between said target cell and said reference cell.

<Desc / Clms Page number 9>

 In step b) of the above method according to the invention, the expression “formation of heteroduplex of the RNA-cDNA type” is intended to denote the formation of heteroduplex of the RNA-cDNA type in a first aliquot containing a given amount of RNA from said reference sample and the formation of heteroduplex of RNA-cDNA type in a second aliquot containing a given amount of RNA from said target sample, preferably the given amounts of RNA contained in the two aliquots are equal.

 In step b) of the above method according to the invention, the formation of said heteroduplexes is carried out by polymerization using a reverse transcriptase and preferably using primers of the oligo dT type, which do not does not preselect any particular type of mRNA. Thus, these heteroduplexes are representative of and reflect the biological reality of the samples considered.

 In step c), the elimination of the mRNA from said heteroduplex mRNA-cDNA formed in step b) is preferably carried out by hydrolysis in the presence of the enzyme RNAse H, an enzyme which does not preselect any particular type d mRNA. Thus, reference cDNA and target cDNA populations representative of the physiological state of the reference cells and of the target cells are obtained.

 In step d), the formation of heteroduplex of the reference mRNA-target cDNA and target mRNA-reference cDNA type from the target and reference cDNA obtained in step c) is obtained by cross addition of an amount of target mRNA and reference mRNA selected in step a), strictly equivalent to the given amount of mRNA of the reference / target sample used in step b).

 The invention preferably relates to a method for separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said non-hybridized RNAs isolated in step e) contain: Unhybridized RNAs isolated from the sample which contained the RNA of the reference sample in step b), overexpressed RNAs or RNAs whose expression is only detectable (or demonstrated) in the cell target relative to the reference cell and, in addition - in the non-hybridized RNAs isolated from the sample having contained the RNA of the target sample in step b), overexpressed RNAs or RNAs of which

<Desc / Clms Page number 10>

 expression is only detectable (or highlighted) in the reference cell compared to the target cell.

Thus, in the method described above, the succession of steps a) to step e) results in producing, from the two reference mRNA and target mRNA samples, four physically distinct samples:
1. a sample containing excess mRNA in the target compared to the reference,
2. a sample containing excess mRNA in the reference relative to the target,
3. a sample containing the target mRNA-reference cDNA hybrids,
4. a sample containing the reference mRNA-target DNA hybrids.

 This physical separation of the different types of mRNA in samples 1 to 4 has the following advantages: i) samples 1 and 2 contain all of the mRNAs which are expressed differently between a target cell and a reference cell, ii) the samples 3 and 4 contain all of the mRNAs which are also expressed in the target cell and reference cell.

 The present method described above, which constitutes the main object of the invention, has the advantage of selecting all of the nucleic acids differently expressed in a reference / target pair. More particularly, the selection of said differently expressed nucleic acids are physically isolated, without any prior amplification step by PCR, cloning, expression in phages, and without any preselection such as hydrolysis by restriction endonucleases.

 In addition, in order to identify differently expressed genes, it seems logical to focus the effort on samples 1 and 2 from said method according to the invention (also called GPI for Gene Profile Identification), and this independently of the technology used for identification (differential display PCR, use of microarrays (such as the Gene ChipTM technology marketed by the company Affymetrix)).

 The invention preferably relates to a method for separating nucleic acid differently expressed between a target cell and a reference cell according to

<Desc / Clms Page number 11>

 the invention, further characterized in that said RNAs hybridized in the form of heteroduplex of RNA-cDNA type isolated in step e) contain RNAs expressed in a similar manner between the target cell and the reference cell.

 By RNAs expressed in a similar manner is intended to denote all of the RNAs expressed by the two said cells and common to these two said cells.

 The invention preferably relates to a separation method according to the invention, characterized in that in step b), the formation of said heteroduplex of cDNA type by polymerization by means of a reverse transcriptase is initiated by oligonucleotides of oligo type (dT), more preferably by oligonucleotides of oligo type (dT) previously labeled with biotin.

 The invention preferably relates to a separation method according to the invention, characterized in that in step b), the cDNA of said heteroduplex of RNA-cDNA type is labeled.

 Among the markers capable of being used for labeling said cDNA heteroduplexes of the RNA-cDNA type, mention may be made in particular, but not limited to, of biotin.

 The invention preferably relates to a separation method according to the invention, characterized in that in step b), the cDNA of said heteroduplex of RNA-cDNA type is labeled with biotin.

 The invention preferably relates to a separation method according to the invention, characterized in that in step c), the elimination of said RNA from said heteroduplex cRNADn formed is carried out by digestion in the presence of RNAse or by alkaline treatment.

 Among the RNAse, there may be mentioned in particular, but not limited to: - RNAse H.

 Preferably, the elimination of said RNA from said RNA-cDNA heteroduplex formed is carried out in step c) by digestion in the presence of RNAse H.

 The invention preferably relates to a separation method according to the invention, characterized in that in step e), the separation of said non-hybridized RNA from said heteroduplex of RNA-cDNA type is carried out in the presence of a solid phase capable to specifically fix said heteroduplex RNA-cDNA and, where appropriate, the cDNAs not hybridized with RNA.

<Desc / Clms Page number 12>

 In a particular embodiment, the invention relates to a separation method according to the invention, characterized in that in step e), the separation of said non-hybridized RNA from said heteroduplex of RNA-cDNA type is carried out by magnetic separation by means of magnetic beads coated with a compound capable of specifically fixing said RNA-cDNA heteroduplex and, where appropriate, cDNAs not hybridized with RNA, preferably said compound is capable of fixing biotin and is chosen from derivatives of avidin such as streptavidin.

 The invention preferably relates to a separation method according to the invention, characterized in that said RNA repertoire originating from said target cell and from said reference cell is a purified fraction of eukaryotic or prokaryotic cell RNA.

 The term “purified RNA fraction” is intended to denote a fraction essentially comprising RNA, devoid in particular of proteins, DNA or RNAase activity capable of interfering with the performance of the method according to the invention.

 Preferably, said purified fraction will be at an RNA concentration greater than or equal to the concentration obtained for an optical density read at 260 nm of 1, that is to say greater than or equal to approximately 40 μg per ml, this fraction can then be diluted to the concentration desired to obtain the given quantity of RNA used in step b) of the method according to the invention.

 Preferably, said target and reference samples chosen are fractions purified at least 90%, more preferably at least 95% and 99% in RNA.

 The invention preferably relates to a separation method according to the invention, characterized in that said repertoire of RNA originating from said target cell and from said reference cell is a purified fraction of mRNA.

 The invention preferably relates to a separation method according to the invention, characterized in that said RNA repertoire originating from said target cell and from said reference cell is a purified fraction of mammalian cell or cell RNA mammalian cell line.

 The invention preferably relates to a separation method according to the invention, characterized in that said RNA repertoire originating from said target cell and from said reference cell is a purified fraction of RNA from human cell or from cell line cell of human origin.

<Desc / Clms Page number 13>

 The invention preferably relates to a separation method according to the invention, characterized in that said repertoire of RNA originating from said target cell is a repertoire of RNA of a tumor cell and in that said repertoire of RNA originating of said target cell is an RNA repertoire of a normal cell.

 In another aspect, the invention also relates to a separation method according to the invention, characterized in that said RNA repertoire originating from said target cell is an RNA repertoire of a cell exhibiting at least one non-tumor character presented by said reference cell.

 Preferably, said at least tumor character presented by said target cell is chosen from the migratory, invasive or proliferative character of a cell.

 The invention preferably relates to a separation method according to the invention, characterized in that said target cell having at least one tumor character is chosen from a metastasis cell, a primary tumor tissue cell, a cell positive for the CG117 marker or an early trophoblast cell.

 The invention preferably relates to a separation method according to the invention, characterized in that said reference cell not presenting at least one tumor character presented by said target cell is chosen from a normal cell, a tumor tissue cell, a CG117 marker negative cell or late trophoblast cell.

 In another aspect, the subject of the present invention is a method of separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said differently expressed nucleic acid is obtained, for each of the samples, in the form of cDNA and in that said method further comprises the following step: f) obtaining cDNA corresponding to said non-hybridized RNAs isolated in step e) by polymerization using reverse transcriptase preferably in the presence of oligo (dT).

 The invention preferably relates to a method of separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said method further comprises the following step: g) elimination of the RNA of the heteroduplex of RNA-cDNA type obtained in step f).

<Desc / Clms Page number 14>

 The invention preferably relates to a method for separating differently expressed nucleic acid between a target cell and a reference cell according to the invention, characterized in that said differently expressed nucleic acid is obtained in the form of double-stranded DNA and in that that said method further comprises a step of polymerizing the cDNA obtained in step f) or g) in the presence of a DNA polymerase.

 In another aspect, the invention relates to a method of separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said method further comprises a step h) of amplification of the cDNA obtained in step f) or g) by chain reaction in the presence of a DNA polymerase.

Xi-X2-X3-...-Xn-oIigo (dN) ou oligo dégénéré pour les amorces sens ; et de type : Yi-Y2-Y3-...-Ym-oligo (dT) pour les amorces retours, dans lesquelles amorces, Xl, X2, X3,..., Xn, YI, Y2, Y3, et Ym représentent de manière indépendante un nucléotide choisi parmi A, T, G ou C, et les nombres n et m étant des entiers supérieurs à zéro, de préférence compris entre 1 et 15. In a particular embodiment, the invention relates to a method of separating nucleic acid differently expressed between a target cell and a reference cell, characterized in that said step h) of amplification of the cDNA obtained at step f) or g) is carried out by a combinatorial type polymerase chain polymerization method (combinatorial PCR) or by a method related to combinatorial PCR in the presence of random oligonucleotide primers of type:

Xi-X2-X3 -...- Xn-oIigo (dN) or degenerate oligo for sense primers; and of type: Yi-Y2-Y3 -...- Ym-oligo (dT) for the return primers, in which primers, Xl, X2, X3, ..., Xn, YI, Y2, Y3, and Ym represent independently a nucleotide chosen from A, T, G or C, and the numbers n and m being integers greater than zero, preferably between 1 and 15.

 Preferably, said number n is equal to 10 and m is equal to 2 or 3.

 Preferably, the oligonucleotide primers used for said combinatorial PCR are labeled, more preferably in the 5'terminal position, in particular in the 5'terminal position of the return primers of oligo (dt) of the combinatorial type.

 Among the markers that can be used for the marking of said random oligonucleotides, preference is given to markers capable of being detected and / or quantified directly or indirectly by a method and / or apparatus making it possible to transform the amount of signal emitted or induced by said marker in a digital signal representative of the quantity of said amplification product obtained, of

<Desc / Clms Page number 15>

préférence par un marqueur de type radioactif, tels que le 32p, ou de type fluorophore (marqueur fluorescent) tels que par exemple les amidites fluorescentes 6F AM, TET, HEX, NED et VIC, les marqueurs fluorescents TAMRA, ROX, Cy3 et Cy5, ou tout autre marqueur connu de l'homme de l'art.

preferably with a marker of radioactive type, such as 32p, or of fluorophore type (fluorescent marker) such as for example the fluorescent amidites 6F AM, TET, HEX, NED and VIC, the fluorescent markers TAMRA, ROX, Cy3 and Cy5, or any other marker known to those skilled in the art.

 In another aspect, the invention also comprises a method of separating nucleic acid differently expressed between a target cell and a reference cell according to the invention, characterized in that said method further comprises at the end of the step h) a step i) of separation of the amplification products obtained for each of said samples, preferably by electrophoresis on polyacrylamide or agarose gel.

 Preferably, said step i) of separation of the amplification products will be followed by a step j) of comparing the nucleic acid bands thus separated for each of said samples and of identifying a nucleic acid band of a nature and / or of different intensity between each of said samples.

 In a preferred embodiment, the separation by electrophoresis and the analysis of the bands or fractions of nucleic acids thus separated will be carried out using a scanner of the FMBiolI (HITACHI) type if the combinatorial oligonucleotide primers are labeled with 1 using flourescent marker.

 Preferably, said step j) of comparing the nucleic acid bands and identification will be followed by a step k) of re-amplification of the PCR products contained in said nucleic acid band of nature and / or intensity different thus identified.

Xi-X2-X3-...-Xn-oligo (dN) ou oligo dégénéré pour les amorces sens ; et de type : YI-Y2-Y3-Y.-oligo (dt) pour les amorces retours, dans lesquelles amorces, Xl, X2, X3,..., Xn, YI, Y2, Y3, et Ym représentent de manière indépendante un nucléotide choisi parmi A, T, G ou C, et les nombres n et m étant des entiers supérieurs à zéro, de préférence compris entre 1 et 15. Preferably, step k) of re-amplification of the PCR products contained in said nucleic acid band of different nature and / or of intensity thus identified will be carried out by combinatorial PCR, in particular by using the pairs of combinatorial primers of type:

Xi-X2-X3 -...- Xn-oligo (dN) or degenerate oligo for sense primers; and of type: YI-Y2-Y3-Y.-oligo (dt) for the return primers, in which primers, Xl, X2, X3, ..., Xn, YI, Y2, Y3, and Ym represent independently a nucleotide chosen from A, T, G or C, and the numbers n and m being integers greater than zero, preferably between 1 and 15.

 Preferably, said number n is equal to 10 and m is equal to 2 or 3.

<Desc / Clms Page number 16>

 Preferably also, said step k) of re-amplification of the PCR products contained in said nucleic acid band of different nature and / or of intensity thus identified can be carried out by a method of PCR type in the presence of a primer drawn at starting from the at least partial sequence of these said PCR products. Thus, such a method according to the present invention will comprise after step j) a step of at least partial sequencing of said PCR products prior to the step of re-amplification of these PCR products.

 In another aspect, the invention relates to a method of cloning and / or expression of a nucleic acid, or of a fragment thereof, differently expressed between a target cell and a reference cell, characterized in what the method comprises the following stages: - obtaining a cDNA, single-stranded or double-stranded, differently expressed between a target cell and a reference cell by a separation method according to the present invention; and - cloning and / or expression of said cDNA in a prokaryotic or eukaryotic cell.

 The cloning and / or expression methods of a cDNA in a cell are today well known to those skilled in the art and will not be developed here. We can for example, but not limited to, use the standard methods explained in the work by Sambrook and A. (1989) cited above.

In another aspect, the subject of the invention is a method of identifying the sequence of a nucleic acid, or of a fragment thereof, expressed differently between a target cell and a reference cell, characterized in that the method includes the following steps:
A) separation and amplification of nucleic acid differently expressed between a target cell and a reference cell by a method according to the present invention; and
B) e sequencing of the sequence of the amplification product obtained at the end of step A).

 Sequencing methods are now well known to those skilled in the art and will not be developed here.

 Among these nucleic acid sequencing methods, there may be mentioned, but not limited to, the Sanger method.

<Desc / Clms Page number 17>

The invention also relates to a method of identifying the sequence of a nucleic acid, or of a fragment thereof, expressed differently between a target cell and a reference cell, characterized in that the method comprises the following steps:
A) separation and amplification of nucleic acid differently expressed between a target cell and a reference cell by a method according to the present invention;
B) sequencing at least one specific fragment of the sequence of the amplification product obtained at the end of step A); and
C) searching in databases for a nucleic sequence comprising the sequence of said at least specific fragment identified in step B), or comprising a sequence having a percentage identity of at least 80%, preferably d '' at least 85%, 90%, 95%, 98% and 99%, with the sequence or the complementary sequence of said at least specific acid fragment identified in step B).

 The term “specific fragment of the sequence of the amplification product obtained in step A) is intended to denote a fragment of said sequence of sufficient length to allow research and identification of the complete sequence from which it is derived, and the case due to its function, in nucleic acid sequence databases.

 Preferably, said specific fragments will have a length of at least 20 consecutive nucleotides of the sequence of the amplification product obtained in step A) of the method according to the present invention, more preferably at least 25.30 , 35, 40.50, 75 or 100 consecutive nucleotides of said sequence.

 By percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is intended to denote a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by comparison window to identify and compare the local regions of sequence similarity. Optimal alignment of sequences for

<Desc / Clms Page number 18>

 comparison can be carried out, in addition to manually, using the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Nut !. Acad. Sci. USA 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).

lesquelles le nucléotide ou le résidu d'acide aminé est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions dans la fenêtre de comparaison et en multipliant le résultat obtenu par 100 pour obtenir le pourcentage d'identité entre ces deux séquences. The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The identity percentage is calculated by determining the number of identical positions for

which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

sequences", FEMS Microbiol Lett. 174 : 247-250) disponible sur le site http ://www. ncbi. nlm. nih. gov/gorf/bl2. html, les paramètres utilisés étant ceux donnés par défaut (en particulier pour les paramètres open gap penaltie : 5, et extension gap penaltie : 2 ; la matrice choisie étant par exemple la matrice BLOSUM 62 proposée par le programme), le pourcentage d'identité entre les deux séquences à comparer étant calculé directement par le programme. For example, we could use the BLAST program, BLAST 2 sequences (Tatusova et al., "Blast 2 sequences-a new tool for comparing protein and nucleotide

sequences ", FEMS Microbiol Lett. 174: 247-250) available on the site http: // www. ncbi. nlm. nih. gov / gorf / bl2. html, the parameters used being those given by default (in particular for parameters open gap penalty: 5, and extension gap penalty: 2; the chosen matrix being for example the BLOSUM 62 matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

 The present invention also comprises a method of identifying a protein, or one of its fragments, encoded by a nucleic acid differently expressed between a target cell and a reference cell, characterized in that the method comprises the following steps : - the identification of said nucleic acid, or of a fragment thereof, by a method according to the invention;

<Desc / Clms Page number 19>

 the identification of said protein, or of one of its fragments, by decoding of said nucleic acid identified in the previous step.

cellule cible et une cellule de référence, caractérisée en ce que la séquence génomique dudit gène comprend la séquence de l'acide nucléique identifié par une méthode d'identification selon l'invention. The present invention also includes a method of identifying a gene encoding a protein, or a fragment thereof, expressed differently between a

target cell and a reference cell, characterized in that the genomic sequence of said gene comprises the sequence of the nucleic acid identified by an identification method according to the invention.

 Preferably, said gene thus identified is a gene coding for a protein involved in the migratory, invasive and / or proliferative nature of a cell such as a tumor cell.

In a last aspect, the subject of the present invention is a method for quantifying the transcription product, or one of its fragments, of a gene expressed differently between a target cell and a reference cell in a given cell, characterized in what the method includes the following steps:
A) the identification of a nucleic acid, or of a fragment thereof, expressed differently between a target cell and a reference cell by a method according to the present invention;
B) the selection and, if necessary, the preparation of a pair of primers or of a probe drawn from said sequence identified in step A); and
C) quantifying said transcription product in said given cell by a nucleic acid quantification method.

 Among the quantification methods, the so-called quantitative PCR methods are preferred, in particular the so-called quantitative real-time PCR methods, or by any quantitative or semi-quantitative PCR technique well known to those skilled in the art. Among the apparatuses based on said detection methods, preference is given in particular to apparatuses based on technologies known as quantitative real-time PCR, such as the so-called Taqman method. Two companies currently supply this type of equipment: PE Biosystems (Poster City CA) for the SDS 7700 and SDS 5700 PLCs and Roche company for the PLC (LIGHT CYCLER). These automata, in equal measure, are capable of detecting fluorescent markers as described above.

<Desc / Clms Page number 20>

The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope.

Legends of the figures: Figure 1: TaqMan curves of quality control TaqMan total RNA versus mRNA. Number of CT amplification cycles as a function of the quantity of mRNA nucleic acid contained in a biological sample. Figure 1 shows 4 Taqman curves &commat; for 4 samples denoted total RNA 1, total RNA 2, messenger RNA 1 and messenger RNA 2 (mRNAs 1 and 2 were prepared from total RNAs 1 and 2), as described in Example 1)). For each of the 4 samples tested, the curve represents the quantity of labeled PCR product obtained for a fragment of 2 microglobulin, expressed in quantity of fluorescence signal detected, as a function of the CT number of amplification cycles carried out at the time of detection.

Figures 2A and 2B. Number of CT amplification cycles as a function of the quantity of nucleic acid contained in an aliquot representative of the various stages of the subtractive hybridization method.

The curve CI represents the relative quantity of cDNA in the form of heteroduplex obtained at the end of step b) of the method according to the present invention.

Curve C2 represents the relative quantity of single-strand cDNA obtained in step c).

Curve C3 represents the relative amount of non-subtracted cDNA present in the supernatant after magnetic separation in step e).

Curve C4 represents the relative amount of non-subtracted cDNA present in the supernatant in step e) after RT-PCR. Thus, C4 includes the relative amount of cDNA corresponding to C3 as well as the relative amount of cDNA from non-subtracted mRNA.

Curve C5 represents the relative amount of subtracted cDNA present on the magnetic solid phase after magnetic separation in step e).

Figure 3. Image of an autoradiography film obtained after the complete development of the subtractive hybridization method, combinatorial PCR, electrophoretic separation in 5% acrylamide gel and autoradiography. The vertical electrophoretic migration bands are grouped by T / NT pairs (Treated cells (T) for target cells, Untreated cells (NT) for reference cell). There are several T / NT pairs corresponding to amplifications by combinatorial PCR with

<Desc / Clms Page number 21>

amorces distinctes Oligo N10 (dégénérées)-SC1, Oligo N10-SC2..... Oligo N10-SC16, selon la description matériels et méthodes. La figure 3 montre de nombreuses bandes distinctes (indiquées par des flèches) correspondant à des ARNm différemment exprimés entre les cellules cibles et les cellules de référence.

separate primers Oligo N10 (degenerate) -SC1, Oligo N10-SC2 ..... Oligo N10-SC16, according to the description of materials and methods. Figure 3 shows many distinct bands (indicated by arrows) corresponding to mRNAs expressed differently between the target cells and the reference cells.

step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 : 156-159, 1987) à l'aide de la solution RNA PLUS&commat; de la société Qbiogene (Appligene). a) A partir d'un échantillon de tissu
On ajoute la solution de lyse RNA PLUS&commat; (2 ml pour 100 mg de tissu), dans chaque tube et l'on termine la lyse en broyant au Thurax pendant quelques secondes. b) A partir d'un échantillon de cellules
Les cellules en suspension sont culottées (par centrifugation), puis lysées par addition de 2 ml de RNA PLUS&commat; pour 10 cellules. EXAMPLE 1 Materials and Methods A Preparation of RNA Extracts from a Sample of Al Cells or Tissue. Preparation of Total RNA Extracts
Total RNA samples are prepared by the method based on cell lysis with guanidine isothiocianate and extraction of RNAs with acid phenol, as described by Chormczinsky and Sachi (Chomczynski, P., and Sacchi, N. Single -

step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987) using RNA PLUS &commat; from the company Qbiogene (Appligene). a) From a tissue sample
Add the RNA PLUS lysis solution &commat; (2 ml per 100 mg of tissue), in each tube and the lysis is ended by grinding with Thurax for a few seconds. b) From a sample of cells
The cells in suspension are pelleted (by centrifugation), then lysed by adding 2 ml of RNA PLUS &commat; for 10 cells.

 For tissue or cell samples, 200 u is then added. I of chloroform in each tube containing 2 ml of lysis solution. Then, the tubes are vigorously agitated (cap closed) with a vortex and they are left to stand for 5 minutes on crushed ice. The following stages of centrifugation, recovery of the aqueous phase, precipitation of the RNA with 1 volume of isopropyl alcohol and rinsing with 70% ethanol, are carried out according to the RNA PLUS protocol &commat; from the manufacturer Qbiogene.

The dry total RNA pellet is taken up in 30 μl of nuclease-free water. The tube is stored in a freezer at a temperature of -20 C
Other possibilities for preparing total RNA can also be used here, such as, but not limited to, methods using columns of ion-exchange resins (Qiagen) or columns comprising a matrix of silicates ( Sigma-Genosys).

<Desc / Clms Page number 22>

A2. Preparation of extracts from messenger RNAs This preparation is made from total RNAs obtained using the Oligotexfg kit) mRNA Mini kit containing the OBB, OW2 and OEB buffers, an Oligotex & commat solution, mini-columns, collecting tubes . The manufacturer's protocol is applied as briefly described below:
The Oligotex solution and the OBB buffer are preheated to 37 C, and the EPO buffer to 70 C.

 The extraction is carried out for a maximum amount of total RNAs of 0.25 mg per column.

- 0, 25 mg d'ARN total dans 250 ul d'eau exempte de nucléase ; - 250 gel de tampon OBB ; - 15 gel d'une solution d'Oligotex&commat; préalablement vortexée (agitée au moyen d'un vortex), pour mettre les billes en suspension. In a 2 ml tube are added:

- 0.25 mg of total RNA in 250 μl of nuclease-free water; - 250 OBB buffer gel; - 15 gel of an Oligotex & commat solution; previously vortexed (agitated by means of a vortex), to put the beads in suspension.

 The tube is mixed by inversion and incubated for three minutes at 70 ° C., then left at room temperature for ten minutes.

 The tube is then centrifuged for two minutes at 14,000 rpm at a temperature of 20C. The supernatant is removed using a Gilson Ph 000 pipette.

 The beads are resuspended in 400 µl of OW2 buffer.

Oligotex&commat;, posée dans un tube collecteur de 1, 5 ml. On centrifuge pendant deux minutes à 14 000 trs/mn à la température de 20C Le tube collecteur est jeté et remplacé par un nouveau. The mixture of beads plus OW2 buffer is placed in a mini-column of the kit

Oligotex &commat;, placed in a collecting tube of 1.5 ml. It is centrifuged for two minutes at 14,000 rpm at a temperature of 20C. The collecting tube is discarded and replaced with a new one.

 In the column, the beads are washed with 400 μl of OW2 buffer with stirring. The column is centrifuged for two minutes at 14,000 rpm at a temperature of 20 C, the collecting tube is discarded and replaced with a new one. This washing step is repeated a second time.

- 20 oil de tampon OEB (préchauffé à 70 C) sont déposés dans la colonne. Les billes sont remises en suspension à l'aide d'une pipette. La colonne est centrifugée pendant The messenger RNAs are eluted from the column in a clean collecting tube with 40 μl of EPO buffer as follows:

- 20 oil of EPO buffer (preheated to 70 C) are deposited in the column. The beads are resuspended using a pipette. The column is centrifuged for

<Desc / Clms Page number 23>

deux minutes à 14 000 trs/mn à la température de 20 C. Cette étape est répétée une fois ; - les 40ul d'éluat sont récupérés et déposés à nouveau dans la colonne. Les billes sont remises en suspension à l'aide d'une pipette. La colonne est centrifugée pendant deux minutes à 14 000 trs/mn à la température de 20 C.

two minutes at 14,000 rpm at a temperature of 20 C. This step is repeated once; - the 40 μl of eluate are recovered and deposited again in the column. The beads are resuspended using a pipette. The column is centrifuged for two minutes at 14,000 rpm at a temperature of 20 C.

B Determination of the purity and the quantity of RNAs in the samples
B 1 Quantity and quality of total RNA
After 24 hours of resuspension at −20 ° C., the concentration of an aliquot of total RNA is measured on a UV spectrophotometer at 260 nm. (Absorbance = elc), taking nuclease-free water as a reference.

 On the other hand, 500 ng of sample are deposited in a 1% agarose gel in the presence of ethidium bromide. After migration, the quality of the total RNA is assessed by visualization in UV illumination of the bands corresponding to the 18S and 28S ribosomal RNAs. The total RNA solution, after aliquoting, is stored at -80 C.

B2. Quantity and quality of messenger RNA:
The concentration of an aliquot of messenger RNA is determined by measurement with a UV spectrophotometer at 260 nm using the EPO buffer for reference.

 On the other hand, similar amounts of total RNA and messenger RNA (250 ng) are deposited on a 1% agarose gel, in the presence of ethidium bromide. The quality and quantity of the messenger RNA are appreciated by the visualization in UV illumination. The mRNA solutions are stored at −20 C.

 Additional quality control is carried out using TaqMan technology (quantitative real-time PCR). A similar amount (expressed in ng / gl) of total RNA and mRNA (250 ng total RNA and 250 ng mRNA) is used for a reverse transcription reaction. The complementary cDNA obtained is used for a quantitative PCR reaction in real time, and for the use of the Taqman ss2 microglobulin probe. The mRNA enrichment is measured, which is expressed by the difference dCT between the number of amplification cycles obtained for a threshold signal quantity value, CT treshold for the curve corresponding to the tRNAs and that corresponding to the mRNAs (see figure 1).

<Desc / Clms Page number 24>

B3 Sampling of the RNA aliquot
The messenger RNAs of the two samples to be studied (reference and target) are adjusted to the same concentration.

 Each type of messenger RNA is aliquoted in two copies of equal volume, one used for reverse transcription, the other (stored at −80 ° C.) for hybridization carried out subsequently.

et des oligo d (T) marqués à la biotine. En zone pré-PCR, sous une hotte à flux vertical, 20 ni de mélange réactionnel RT sont préparés, dans des tubes exempts de nucléases de 1, 5 ml à vis. C) Formation of RNA-cDNA heteroduplexes
The two messenger RNAs are transcribed with a reverse transcriptase enzyme

and biotin-labeled oligo d (T). In the pre-PCR zone, under a vertical flow hood, 20 μl of RT reaction mixture are prepared, in 1.5 ml nuclease-free tubes with screws.

This reaction mixture is composed per tube of: - 2 μl of 10 mM dNTP (Pharmacia); - 8 gel of 5X buffer (Gibco BRL); - 4 O of DTT at 0.1 M (Gibco BRL); - 4 t of oligo d (T) labeled with biotin at 29 μM (Genset); -2 ph of Reverse Transcriptase Super Script II enzyme at 200 U / pl (Gibco BRL).

This reaction mixture is used to transcribe 20 µl of messenger RNA. A negative reverse transcription control is obtained by replacing the messenger RNA with nuclease-free water. The messenger RNAs are added outside the hood, in the pre-PCR zone.

 The synthesis is carried out by incubating the tubes for ten minutes at 23 ° C. and one hour at 42 ° C.

D) Elimination of RNA from heteroduplex RNA-cDNA
Once the cDNA synthesis is complete, 2 μl of RNase H + enzyme at 3.4 U / l are added to the reaction mixture in order to degrade the messenger RNAs and incubated for thirty minutes at 37 C. The RNase H + enzyme is then denatured by heating at 85 C for ten minutes.

 At this stage, a sample is taken (Control 1) to monitor the progress of the samples and determine the quality of the manipulation: 1 μm each cDNA (target and reference) is diluted in 19 nuclease-free water gel. The Control 1 target cDNA and Control 1 reference cDNA tubes are stored at −20 C.

<Desc / Clms Page number 25>

Purification of reverse transcription No. 1 is carried out in 2 ml round bottom tubes. The target and reference cDNAs are precipitated to remove the excess oligo d (T) with 1 / 10th of a volume of a 3M sodium acetate solution, pH 4.8 and 2.5 volumes of absolute ethanol, in the following conditions: - incubation for 30 minutes at -20 C, then centrifugation at 14,000 rpm (at +4 C) for thirty minutes; - the supernatants are removed by suction and the pellets are rinsed with 500 μl of 70% ethanol (cooled to -20 ° C.) by centrifuging at 14,000 rpm (at +4 ° C.) for ten minutes; - the supernatants are removed by suction and the pellets dry in the open air for 2 minutes.

E) Formation of the RNA-cDNA heteroduplex from the cDNAs obtained in D) The hybridizations are carried out by mixing the cDNA obtained previously with the conserved messenger RNA from the other sample (cross-mixture).

 The target and reference messenger RNAs stored at −80 ° C. are thawed. The target and reference cDNA pellets resulting from the purification of reverse transcription No. 1 are taken up in 20 μl of hybridization buffer and 2 μl of RNase inhibitor are added.

 The resuspended cDNA is transferred into a 0.2 ml PCR tube then: - to the 20 μl of the target cDNA are added 20 μl of reference messenger RNA, - to the 20 μl of the reference cDNA are added 20 μl d 'Target messenger RNA.

 The hybridizations are carried out in a thermocycler which makes it possible to obtain a regular temperature gradient between 70 ° C. and 4 ° C., the temperature falling by 5.5 ° C. every hour for 12 hours.

pour suivre l'évolution des échantillons et déterminer la qualité de la manipulation : 1 gel de chaque hétéroduplex est dilué dans 19 ul d'eau exempte de nucléase. Les tubes Contrôle 2 ADNc cible et Contrôle 2 ADNc référence sont stockés à-20 C. At the end of the hybridizations a second sample (Control 2) is carried out

to follow the evolution of the samples and determine the quality of the manipulation: 1 gel of each heteroduplex is diluted in 19 μl of nuclease-free water. The Control 2 target cDNA and Control 2 reference cDNA tubes are stored at −20 C.

F) Separation of the non-hybridized RNA from the RNA-cDNA heteroduplex obtained in E) The heteroduplex solutions obtained in E) contain a mixture composed of biotin-labeled excess cDNA, of mRNA-cDNA complexes (labeled with biotin), and excess mRNA. Separation is carried out using microbeads

<Desc / Clms Page number 26>

 magnetic carriers of streptavidin, which has an affinity for biotin. The attachment to the streptavidin beads of any material labeled with biotin makes it possible to recover from the supernatant only the excess mRNA.

le tube par l'action d'un aimant (ce qui permet la séparation billes/surnageant), puis 150 III de tampon B & W (2X) sont ajoutés aux billes. Ce tampon est éliminé de la même façon. L'étape soustractive est réalisée en tampon B & W (IX). Un deuxième lavage des billes est effectué avec ce tampon, en diluant au demi le tampon B & W (2X) par de l'eau exempte de nucléase (75 III d'eau exempte de nucléase pour 75 III de tampon B & W (2X)). The procedure is as follows:
150 III of streptavidin beads are distributed in four 1.5 ml tubes. The beads are first washed in a B & W buffer (2X) and the bead storage solution is removed by pipetting, the magnetic beads being retained in

the tube by the action of a magnet (which allows the separation of the beads / supernatant), then 150 III of B & W buffer (2X) are added to the beads. This buffer is eliminated in the same way. The subtractive step is carried out in B & W (IX) buffer. A second washing of the beads is carried out with this buffer, diluting the B & W buffer (2X) by half with nuclease-free water (75 III of nuclease-free water for 75 III of B & W buffer (2X )).

In a first tube containing the washed beads, are added: - 40 μl of the hybridization solution containing the heteroduplexes, -40 ph of B & W buffer (2X).

The tube is then incubated for 10 min at room temperature, then 20 min at 37 C.

In a second tube containing the washed beads, the 80 III of supernatant recovered from this first tube is added using a magnet. This second tube is incubated for 10 min at room temperature, then 20 min at 37 C. The supernatant containing the excess mRNA is removed using a magnet and transferred to 2 ml tubes.

 The beads which have fixed the heteroduplexes and the excess cDNA are resuspended in 40 μl of buffer.

 On the supernatants of the two samples, a third sample (Control 3) is carried out to follow the evolution of the samples and to determine the quality of the handling as follows: - 2 III of each subtraction are diluted in 18 μl of free water nuclease. The Control 3 target cDNA (reference excess mRNA) and Control 3 reference cDNA (target excess mRNA) tubes are stored at −20 C.

 The mRNA contained in the supernatants of the two subtractions is precipitated with 1 / 10th of a 3M sodium acetate solution, pH 4.8 and 2.5 times the volume in absolute ethanol, under the following conditions:

<Desc / Clms Page number 27>

- incubation trente minutes à-20 C, puis centrifugation à 14 000 trs/mn (à +4 C) pendant trente minutes ; - les surnageants sont éliminés par aspiration et les culots sont rincés avec 500 ul d'éthanol à 70 %, refroidi à-20 C en centrifugeant à 14 000 trs/mn (à +4 C) pendant dix minutes ; - les surnageants sont éliminés et les culots sèchent à l'air libre pendant 2 minutes.

- thirty minute incubation at -20 C, then centrifugation at 14,000 rpm (at +4 C) for thirty minutes; - the supernatants are removed by suction and the pellets are rinsed with 500 μl of 70% ethanol, cooled to -20 ° C. by centrifuging at 14,000 rpm (at +4 ° C.) for ten minutes; - the supernatants are eliminated and the pellets dry in the open air for 2 minutes.

- 2 al d'une solution d'hexamère aléatoire ( random hexamer ) à 50 uM (PE Biosystems) ; - 1 al d'enzyme RNase inhibitor à 20 U/lll (PE Biosystems) ; d'enzyme Reverse Transcriptase à 200 U/ (Gibco BRL) ; - 2 ul d'eau exempte de nucléase. Then the excess target mRNA pellet and the excess reference mRNA pellet are taken up in 20 u) of RT reaction mixture (Reverse Transcriptase): - 2 g of 10 mM dNTP (Pharmacia); - 8 gel of 5X buffer (Gibco BRL); - 4 gel of 0.1 M DTT (Gibco BRL);

- 2 μl of a 50 μM random hexamer solution (PE Biosystems); - 1 al of RNase inhibitor enzyme at 20 U / lll (PE Biosystems); 200 U / Reverse Transcriptase enzyme (Gibco BRL); - 2 μl of nuclease-free water.

This reaction mixture is prepared in a 1.5 ml screw tube under the hood and added to the two dry pellets of excess mRNA. Reverse transcription is carried out by incubating the tubes for ten minutes at room temperature and one hour at 42 ° C., then the enzymes are denatured at 100 ° C. (boiling) for ten minutes.

At this stage, a fourth sample (Control 4) is carried out to monitor the progress of the samples and determine the quality of the handling as follows:
1 µm die each reaction mixture of reverse transcription is diluted in 19 nuclease-free water gel. The Control 4 cDNA tubes of the reference excess mRNA and the Control 4 cDNA of the target excess mRNA are stored at −20 C.

The streptavidin beads, resuspended after subtraction in 40 gel buffers, are incubated at 100 ° C. for 1 hour. It is from the supernatant that the fifth and last control is carried out (Control 5) as follows: - 1 μl of supernatant is diluted in 19 μl of nuclease-free water. The Reference 5 Control cDNA and Target 5 Control cDNA tubes are stored at −20 C.

<Desc / Clms Page number 28>

G) combinatorial PCR
The reference and target excess cDNAs to be amplified are diluted 1/5 with nuclease-free water to carry out combinatorial PCR.

- 0, 25 ul d'enzyme Hot Start à 5 U/ - 2 oil amorce F (sens) (deglO) à 10 pmol/ - 2 al amorce R (retour) (SC1 à 16 marqués au y33 P) à 10 pmol/ul. The reaction mixture of 17.5 μl is composed per tube of: - 9.25 μl of nuclease-free water; - 2 l of Hot Start 10X buffer; - 2 l of 2 mM dNTP (A, T, C, G);

- 0.25 μl of Hot Start enzyme at 5 U / - 2 oil primer F (sense) (deglO) at 10 pmol / - 2 al primer R (return) (SC1 to 16 labeled with y33 P) at 10 pmol / ul.

The primers SC1 to SC16 correspond to the formula Y1-Y2-18 dT.

17.5 µl of this reaction mixture is used to amplify 2.5 µl of excess cDNA diluted 1/5. Each cDNA is amplified with the 16 pairs of primers. A negative PCR control is added, in which the 2.5 μl of cDNA are replaced by the same volume of nuclease-free water.

 The thermocycler's program is as follows: - 96C for 10 minutes; - 95C for 1 minute, 46 C for 1 minute, 72 C for 1 minute: 40 cycles; - 72C for 10 minutes.

H) Polyacrylamide gel electrophoresis (Differential Display) for the detection of cDNA bands corresponding to RNAs differently expressed
A 5% acrylamide electrophoresis gel is prepared between 2 glass plates.

 The plates are installed in the vertical electrophoresis chamber, migration buffer (TBE IX) is added to the upper and lower cells. Preheating to 45 C of the gel is necessary before depositing.

- 0, 4 ul de bleu 10X ; - 1, 2 ul de formamide ; - 2 l d'échantillon. The samples are prepared for deposit in a 96-well plate with per well:

- 0.4 μl of 10X blue; - 1.2 μl of formamide; - 2 l of sample.

<Desc / Clms Page number 29>

 The entire mixture is deposited on the gel. The amplicons coming from the two samples to be compared made with the same primer pair are placed side by side. Migration takes place at 3000 V (74 W) for 3 hours.

 The gel is transferred to Whatman paper, and dried. Then the gel covered with a film is placed in an autoradiography cassette. On the film and on the paper with the gel, marks are traced, in order to allow the location of the film after development, after 24 h of autoradiography.

 After development, the film and the gel are superimposed, then portions of gel corresponding to the PCR bands visualized on the film are cut. The gel strips are dissolved in 50 u. l of water. The small amount of material thus removed is amplified by PCR using the pair of primers used during the first combinatorial PCR.

- 2 ul de tampon Hot Start 10X ; - 2 ul de dNTP (T) à 2 mM ; - 0, 25 ul d'enzyme Hot Start à 5 U/, ul ; - 2 gel amorce F (deglO) à 10 pmol/ul ; - 2 ni amorce SC à 10 pol/ Ce mélange réactionnel est utilisé pour amplifier les 3 ul d'ADN prélevé dans la bande d'intérêt. Un témoin négatif de PCR est ajouté, où les 3 ul d'ADN sont remplacés par le même volume d'eau exempte de nucléase. I) Combinative PCR and / or sequencing of the cDNAs corresponding to the differently expressed RNAs demonstrated by gel electrophoresis (Differential Display)
In the pre-PCR zone, under a vertical flow hood, 17 l of reaction mixture is produced in 0.2 ml tubes: -8.75 l of nuclease-free water;

- 2 μl of Hot Start 10X buffer; - 2 μl of 2 mM dNTP (T); 0.25 μl of Hot Start enzyme at 5 U / μl; - 2 gel primer F (deglO) at 10 pmol / μl; - 2 ni SC primer at 10 pol / This reaction mixture is used to amplify the 3 μl of DNA taken from the band of interest. A negative PCR control is added, where the 3 µl of DNA is replaced with the same volume of nuclease-free water.

 The tubes are deposited on the thermostatic block of the thermocycler.

 The thermocycler's program is as follows: - 96C for 10 minutes; - (95 C for 1 minute, 46 C for 1 minute): 40 cycles; - 72C for 10 minutes.

<Desc / Clms Page number 30>

J) Quantitative PCR carried out to evaluate the relative amounts of cDNA present for each of the stages tested (Curves C1, C2, C3, C4 and C5) Summary of the samples: - Control 1 target cDNA; - Control 1 reference cDNA; - Control 2 target cDNAs; - Control 2 reference cDNA; - Control 3 target cDNA (reference excess mRNA); - Control 3 reference cDNA (target excess mRNA); - Control 4 cDNA of reference excess mRNA; - Control 4 target excess mRNA cDNA; - Control 5 target cDNA; and - Control 5 reference cDNA.

The samples do not require any special preparation, no dilution is necessary.

1 eye of each control will be used for quantitative RT-PCR.

The samples are quantified, using the microglobulin ss2 probe, proportional to the amount of mRNA present in the sample.

In the pre-PCR zone, under a vertical flow hood, 20 μl of RT-PCR reaction mixture are prepared from the "TaqMan PCR Universal Master Mix" kit, in 0.2 ml Optical tubes "TaqMan" (PE Biosystems ).

The reaction mixture of 20 μl is composed per tube of: - 5 gel of nuclease-free water; - 12.5 μl of Universal Master Mix; - 0.5 μl ss2 probe 10 mM microglobulin; - 1 l primer F ss2 10 mM microglobulin; and - 1 l primer R ss2 10 mM microglobulin This reaction mixture is used to quantify the cDNA present in 5 μl of each control. The tubes are closed with caps in Optical "TaqMan" type bars, and are placed on the thermostatic block of the "7700 Sequence Detector System" (PE Biosystems) device.

The thermocycler program is as follows:

<Desc / Clms Page number 31>

 - 50C for 2 minutes; - 95C for 10 minutes; - (95 C for 15 seconds, 60 C for 1 minute) x 40 cycles.

 The data of each control are compared, which makes it possible to follow the evolution of the mRNA concentrations during the analytical process.

EXAMPLE 2 Results and Interpretation 1 The results in FIG. 1 represent the quantity of signal obtained as a function of the number of CT amplification cycles for the 4 samples tested, denoted total RNA 1, total RNA 2, messenger RNA 1 and messenger RNA 2 (mRNAs 1 and 2 were prepared from total RNAs 1 and 2 respectively) and thus make it possible, for a given quantity of threshold signal, to compare here the quantities of RNA (total and / or messengers) initially contained in the samples tested.

 The dCT1 value representing the difference, expressed in number of amplification cycles for a threshold signal quantity, between the quantity of messenger RNA ss2 microglobulin contained in 1 lag of total RNA 1 and that contained in 1 u. g of a messenger RNA preparation 1 obtained from the total RNA sample 1, is here 7.6.

 The dCT2 value representing the difference between the amount of messenger RNA ss2 microglobulin contained in 1 u. g total RNA 2 and that contained in 1 log a preparation of messenger RNA 2 obtained from the sample total RNA 2, is here 7.56.

The microblobulin ss2 probe can be considered as representative for all of the mRNAs in the biological sample.

The 2 mRNA samples are of very similar quality (same enrichment in mRNA from the total extract, dCT1 = dCT2).

 On the other hand, the total RNA samples 1 and 2, supposed to have the same RNA concentration, determined for example by optical densitometry and the absorbance at 260 nm, can be if necessary strictly rebalanced at the same concentration from the present technique. Taqman &commat; (to obtain two perfectly superimposable curves), a technique which is much more precise and will thus allow

<Desc / Clms Page number 32>

 to perfectly balance the amounts of total RNA 1 and 2, and messenger RNA 1 and 2, for the following steps of the method according to the present invention.

 II FIG. 2A represents the quantity of signal obtained as a function of the number of CT amplification cycles for the 5 control samples CI to C5 in which the target and initial reference sample has a similar quantity of mRNA for ss2 microglobulin. The equation CI = C2 (very similar, almost superimposable curves)) and C3 = C4 (also very similar) demonstrate the effectiveness of the subtractive method.

l'échantillon cible contient un excès d'ARNm ss2 microglobuline par rapport à la référence. L'équation CI = C2 (très similaires) et C4 C3 (C4 très supérieur à C3) démontrent également l'efficacité de la méthode. L'échantillon C4 ainsi sélectionné et séparé contient les ARNm différemment exprimés entre l'échantillon cible et de référence. Figure 2B represents the quantity of signal obtained as a function of the number of CT amplification cycles for the 5 control samples CI to C5 in which

the target sample contains an excess of microglobulin ss2 mRNA compared to the reference. The equation CI = C2 (very similar) and C4 C3 (C4 much higher than C3) also demonstrate the effectiveness of the method. The C4 sample thus selected and separated contains the mRNAs expressed differently between the target and reference samples.

 III FIG. 3 represents the image of the autoradiography film obtained after the complete progress of the subtractive hybridization method, combinatorial PCR, electrophoretic separation in 5% acrylamide gel and autoradiography. The electrophoretic migration bands grouped by T / NT pairs (Treated cells (T) for target cells, Untreated cells (NT) for reference cell) show many distinct bands (more than 86 distinct bands on the original gel), bands distinct corresponding to differently expressed mRNAs between the target cells and the reference cells.

Claims (35)

 1 / Method for separating nucleic acid differently expressed between a target cell and a reference cell, characterized in that said method comprises the following steps: a) the selection of a first target sample and a second reference sample , which samples respectively contain a repertoire of RNA from said target cell and said reference cell; b) the formation of heteroduplex of RNA-cDNA type from a given quantity of RNA contained in said reference sample by polymerization by means of reverse transcriptase; c) removing the RNA from said RNA-cDNA heteroduplex formed in step b); d) the formation of heteroduplex of RNA-cDNA type from the cDNA obtained in step c) by adding an amount of RNA from the target sample selected in step a) equivalent to the amount reference sample RNA data used in step b); e) separation of the RNA added in step d) not hybridized from the heteroduplexes of the RNA-cDNA type obtained in step d); the non-hybridized RNAs isolated in step e) containing the RNAs expressed differently between said target cell and said reference cell.  2 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to claim 1, characterized in that said non-hybridized RNAs isolated in step d) contain overexpressed RNAs or RNAs including expression is only detectable in the target cell compared to the reference cell.  3 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to claim 1 or 2, characterized in that said method comprises the following steps: a) the selection of a first target sample and d a second reference sample, which samples respectively contain a repertoire of target mRNA and reference mRNA originating from said target cell and from said reference cell; <Desc / Clms Page number 34>  b) the formation of heteroduplex of the reference mRNA-reference cDNA from a given quantity of reference mRNA contained in said reference sample and the formation of heteroduplex of the target mRNA-target cDNA from a quantity target mRNA data contained in said target sample by polymerization using reverse transcriptase; c) removing the mRNA from said mRNA-cDNA heteroduplex formed in step b) for each of the samples obtained in step b); d) the formation of heteroduplex of mRNA-cDNA type from the cDNA contained in the sample for each of said samples obtained in step c) by addition: - in the sample obtained in step c) containing the cDNA corresponding to the mRNA of the target sample, of an amount of mRNA of the reference sample selected in step a) equivalent to the given amount of mRNA of the target sample used in l step b), and - in the sample obtained in step c) containing the cDNA corresponding to the mRNA of the reference sample, of an amount of mRNA of the target sample selected in the step a) equivalent to the given quantity of mRNA of the reference sample used in step b); e) the separation for each of the samples obtained in step d) of the mRNA added in step d) not hybridized from the heteroduplexes of mRNA-cDNA type obtained in step d); the non-hybridized mRNAs isolated in each of the samples in step e) containing the mRNAs expressed differently between said target cell and said reference cell.  4 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to claim 3, characterized in that said non-hybridized RNAs isolated in step e) contain: - in non-hybridized RNAs isolated from starting from the sample having contained the RNA of the reference sample in step b), overexpressed RNAs or RNAs whose expression is only detectable in the target cell compared to the reference cell and, in in addition - in the non-hybridized RNAs isolated from the sample which contained the RNA of the target sample in step b), overexpressed RNAs or RNAs whose expression is only detectable in the reference cell by report to the target cell. <Desc / Clms Page number 35>  5 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to one of claims 1 to 5, further characterized in that said RNAs hybridized in the form of heteroduplex of cDNA type isolated at step e) contain RNAs expressed in a similar manner between the target cell and the reference cell.  6 / separation method according to one of claims 1 to 5, characterized in that in step b), the formation of said heteroduplex of RNA-cDNA type by polymerization by means of a reverse transcriptase is initiated by oligonucleotides of oligo type (dT).  7 / separation method according to one of claims 1 to 6, characterized in that in step b), the cDNA of said heteroduplex of RNA-cDNA type is labeled 8 / separation method according to claim 7, characterized in that in step b), the cDNA of said heteroduplex of RNA-cDNA type is labeled with biotin.  9 / separation method according to one of claims 1 to 8, characterized in that in step c), the elimination of said RNA from said heteroduplex RNA-cDNA formed is carried out by digestion in the presence of RNAse or by alkaline treatment .  10 / separation method according to claim 9, characterized in that the RNAse is an RNAse H.  11 / separation method according to one of claims 1 to 10, characterized in that in step e), the separation of said non-hybridized RNA from said heteroduplex of RNA-cDNA type is carried out in the presence of a solid phase capable to specifically fix said heteroduplex RNA-cDNA and, where appropriate, cDNAs not hybridized to RNA 12 / Method of separation according to claim 11, characterized in that in step e), the separation of said non-hybridized RNA from said heteroduplex of cDNA type is carried out by magnetic separation by means of magnetic beads coated with a compound capable of fixing specifically said RNA-cDNA heteroduplexes and, where appropriate, cDNAs not hybridized to RNA 13 / separation method according to claim 12, characterized in that said magnetic beads are coated with a compound capable of fixing biotin, preferably streptavidin. <Desc / Clms Page number 36>  14 / A separation method according to one of claims 1 to 13, characterized in that said RNA repertoire derived from said target cell and from said reference cell is a purified fraction of eukaryotic or prokaryotic cell RNA.  15 / separation method according to claim 14, characterized in that said RNA repertoire from said target cell and said reference cell is a purified fraction of mRNA.  16 / separation method according to one of claims 14 and 15, characterized in that said RNA repertoire from said target cell and from said reference cell is a purified fraction of mammalian cell or cell RNA mammalian cell line.  17 / A separation method according to claim 16, characterized in that said RNA repertoire derived from said target cell and from said reference cell is a purified fraction of RNA from human cell or from cell line cell of human origin .  18 / separation method according to one of claims 16 and 17, characterized in that said RNA repertoire from said target cell is an RNA repertoire from a tumor cell and in that said RNA repertoire from of said reference cell is an RNA repertoire of a normal cell.  19 / A separation method according to one of claims 16 and 17, characterized in that said repertoire of RNA from said target cell is a repertoire of RNA of a tumor cell having at least one tumor character not presented by said reference cell.  20 / separation method according to claim 19, characterized in that said at least tumor character presented by said target cell is chosen from the migratory, invasive or proliferative character of a cell.  21 / separation method according to claim 20, characterized in that said target cell having at least one tumor character is chosen from a metastasis cell, a tumor tissue cell, a cell positive for the CG117 marker or an early trophoblast cell .  22 / separation method according to one of claims 19 to 21, characterized in that said reference cell not having at least one tumor character presented by said target cell is chosen from a normal cell, a cell of <Desc / Clms Page number 37>  tumor tissue, a CG117 marker negative cell or a late trophoblast cell 23 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to one of claims 1 to 22, characterized in that said differently expressed nucleic acid is obtained in the form of cDNA and in that said method further comprises the following step: f) obtaining cDNA corresponding to said non-hybridized RNAs isolated in step e) for said or each of said samples, by polymerization using a reverse transcriptase, preferably by presence of oligo (dT).  24 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to claim 23, characterized in that said method further comprises the following step: g) elimination of the RNA from the 'heteroduplex of RNA-cDNA type obtained in step f) for said or each of said samples.  25 / A method of separating nucleic acid differently expressed between a target cell and a reference cell according to claim 23 or 24, characterized in that said method further comprises a step h) of amplification of the cDNA obtained at l step f) or g) by chain reaction in the presence of a DNA polymerase for said or each of said samples.  26 / A method of separation of nucleic acid differently expressed between a target cell and a reference cell according to claim 25, characterized in that said step h) of amplification of the cDNA is carried out by a method of chain polymerization with combinatorial type polymerase (combinatorial PCR), or by a method related to combinatorial PCR, in the presence of random oligonucleotide primers of type:

 Xi-X2-X3 -...- Xn-oIigo (dN) or degenerate oligo for sense primers; and of type: YI-Y2-Y3 -...- y m-oligo (dT) for the return primers, in which primers, Xl, X2, X3, .., Xn, YI, Y2, Y3, and Ym represent independently a nucleotide chosen from A, T, G or C, and the numbers n and m being integers greater than zero, preferably between 1 and 15.  27 / A nucleic acid separation method according to claim 26, characterized in that said number n is equal to 10 and m is equal to 2 or 3. <Desc / Clms Page number 38>  28 / A method of nucleic acid separation according to claim 26 or 27, characterized in that said method further comprises at the end of step h) a step i) of separation by gel electrophoresis of the amplification products obtained for each of said samples.  29 / A method of nucleic acid separation according to claim 28, characterized in that said method further comprises at the end of step i), a step j) of comparing the nucleic acid bands thus separated for each of said samples and identification of a nucleic acid band of different nature and / or intensity between each of said samples.  30 / A method of nucleic acid separation according to claim 29, characterized in that said method further comprises at the end of step j), a step k) of re-amplification of the PCR products contained in said band of nucleic acid of different nature and / or intensity thus identified.  31 / Method for identifying the sequence of a nucleic acid, or of a fragment thereof, expressed differently between a target cell and a reference cell, characterized in that the method comprises the following steps: A) separation and amplification of nucleic acid differently expressed between a target cell and a reference cell by a method according to one of claims 25 to 30; and B) sequencing the sequence of the amplification product obtained at the end of step A).  32 / Method for identifying the sequence of a nucleic acid, or of a fragment thereof, expressed differently between a target cell and a reference cell, characterized in that the method comprises the following steps: A) separation and amplification of nucleic acid differently expressed between a target cell and a reference cell by a method according to one of claims 25 to 27; B) sequencing at least one specific fragment of the sequence of the amplification product obtained at the end of step A); and C) searching in databases for a nucleic sequence comprising the sequence of said at least specific fragment identified in step B), or comprising a sequence having a percentage identity of at least 80% with the sequence <Desc / Clms Page number 39>  or the complementary sequence of said at least specific fragment identified in step B).  33 / Method for identifying a protein, or one of its fragments, encoded by a nucleic acid differently expressed between a target cell and a reference cell, characterized in that the method comprises the following steps: - identifying said nucleic acid, or a fragment thereof, by a method according to claim 31 or 32; the identification of said protein, or of one of its fragments, by decoding of said nucleic acid identified in the previous step.  34 / Method for identifying a gene coding for a protein, or a fragment thereof, expressed differently between a target cell and a reference cell, characterized in that the genomic sequence of said gene comprises the sequence of the nucleic acid identified by a method according to one of claims 31 and 32.  35 / Method for quantifying the transcription product, or one of its fragments, of a gene expressed differently between a target cell and a reference cell in a given cell, characterized in that the method comprises the following steps: A) identifying a nucleic acid, or a fragment thereof, expressed differently between a target cell and a reference cell by a method according to claim 31 or 32; B) the selection and, if necessary, the preparation of a pair of primers or of a probe drawn from said sequence identified in step A); and C) quantifying said transcription product in said given cell by a nucleic acid quantification method.