FR2874026A1 - QUANTIFICATION OF PROTEIN INTEREST MRIAS USING A REAL-TIME RT-PCR REACTION - Google Patents

Abstract

The present invention relates to the field of the quantification of RNAs in a biological sample, in particular the quantification of messenger RNAs encoding cytokines. The present invention allows absolute quantification of the levels of messenger RNAs of human cytokines present in a biological sample, by a real-time RT-PCR technique based on the use of highly specific primers, the detection of amplifications by incorporation of 'a fluorescent intercalator, and absolute quantification using internal RNA standards (housekeeping genes) and external (standard RNA reference range). The present invention relates to the simultaneous quantification of different RNAs, under the same experimental conditions.

Description

The present invention relates to the field of RNA quantification

  in a biological sample, in particular the quantification of messenger RNAs coding for cytokines. The present invention relates to the simultaneous quantification of different RNAs, in the same

experimental conditions.

  The quantification of messenger RNAs and viral RNAs is a potential tool of great interest, both in the field of research and in the clinical field. In particular, cytokine-specific messenger RNA levels represent highly sensitive markers of the activation state of the immune system in vivo. In the field of research, knowledge of these rates in different models may therefore allow a better knowledge of the immune system. Of course, knowledge of these rates is also crucial information for many clinical situations. Knowing precisely the level of expression of this or that cytokine, can indeed be an important parameter for the management of patients suffering from inflammatory, infectious or immune diseases. An exemplary application of the quantification of messenger RNAs encoding certain cytokines to assess a disease state in a patient with rheumatoid arthritis is described in US Patent 6,555,320.

  One of the means that can be used to quantify messenger RNAs is RT-PCR. RT-PCR is an acronym commonly used in molecular biology, for reverse transcription polymerase chain reaction, or reverse transcription followed by polymerase chain reaction. This type of reaction therefore comprises two steps: a reverse transcription step, that is to say, a synthesis of a single-stranded DNA complementary to an RNA sequence, and a polymerase chain amplification step. The first is here referred to as either the reverse transcription or the English acronym RT. Similarly, the second phase will be designated either by the term polymerase chain reaction, or by the French acronym ACP, or by the English acronym PCR, more commonly used by biologists.

  Compared to the detection of the corresponding cytokines in biological fluids or immunohistochemically cut, the detection of messenger RNAs by RT-PCR is a much more sensitive technique allowing the detection in very small samples, such as biopsies. skin. Other messenger RNA detection techniques, such as RnaseProtectionAssay (RPA Kits, BDBiosciencesTM) and Northern Biot techniques (QuantikineTM mRNA, R & DTM) sometimes require more than 10 μg total RNA for detection of a single cytokine, quantity that is not always reached for many clinical samples, especially in biopsies. In addition to the need for a large amount of starting material, these techniques are very slow to implement and may involve the use of radioactivity.

  RT-PCR is a very sensitive technique, since it makes it possible to achieve a significant increase in the initial number of targets. It is easier and faster to implement. However, very small variations in the efficiency of RT and / or PCR can very significantly affect the end result. It is therefore necessary to control both the RT and PCR phases. Conventional techniques that are based on plateau RT-PCR do not allow absolute quantification because the results are acquired during the saturation phase. Competitive techniques, where the target is co-amplified and an internal standard with the same efficiency, overcome this disadvantage, but have a lower sensitivity due to the co-amplification of two distinct sequences. As a result, they require large amounts of equipment, and can only be implemented for target / competitor ratios of limited extent (generally between 1:10 and 10: 1). In addition, pre-screening is generally necessary to estimate the order of magnitude in which the amount of transcript to be detected is located. Finally, the analysis of the results is long and tedious (Bustin 2000).

  Real-time PCR techniques have opened the way to absolute quantification (Bustin 2000, Ginzinger 2002). Indeed, the detection of the amplification product is done in real time (hence their name) through the use of fluorescent markers. The quantification of the transcripts is done during the exponential phase, the only phase where the amount of amplicon is only dependent on the initial quantity of target. The addition of an external standard then allows absolute quantification if it is amplified with the same efficiency. This technique is also easier to automate, and the analysis of the results relatively simple. This explains why it is more and more used despite a high cost (Bustin 2000, Giulietti, Overbergh et al., 2001).

  Cytokine assays using RT-PCR in real time use mostly fluorescent probes (TagmanTM, molecular beaconTM, etc.) to detect amplicon (Stordeur, Poulin et al., 2002). This approach is interesting because only the desired amplicon is detected despite possible non-specific amplifications. However, the use of these fluorescent probes results in a significant additional cost. In addition, the lower requirement on the specificity of the primers has significant potential biases in the quantization. Indeed, in cases where the primers are not totally specific, several amplifications are likely to be performed in competition. Moreover, these techniques all use a standard DNA range, and therefore do not take into account, in their quantification, the efficiency of the reverse transcription phase. Reverse transcription, however, has a variable efficiency, as shown in Example 1.2 below. As a result, messenger RNA quantification based on a real-time RT-PCR reaction using as an external standard a range of DNA can not claim to be absolute quantitation.

  The object of the present invention is to overcome at least in part the disadvantages mentioned above, and to allow absolute quantification of the determined levels of messenger RNAs, and in particular messenger RNAs of human cytokines present in a biological sample.

  For this, the invention uses a real-time RT-PCR technique based on the use of highly specific primers, the detection of amplifications by incorporation of a fluorescent intercalant, and the absolute quantification by means of RNA standards. internal (housekeeping genes) and external (standard reference RNA range).

  The present invention therefore relates to a method for quantifying one or more determined RNAs, especially at least two distinct RNAs, in a biological sample, comprising the following steps: (i) for each of the RNAs to be quantified, reverse transcription, and then amplification of the product of this reverse transcription, using a specific pair of primers of said RNA, from the biological sample, and from a range of synthetic RNA comprising the sequence amplified by the couple specific primers, (ii) real-time product detection of the amplification reactions of step (i), and (iii) deduction of the amount of RNA in the sample.

  In a particular embodiment, the invention relates to a method of absolute quantification of one or more target RNAs in a biological sample, comprising the following steps: for each of the target RNAs to be quantified, reverse transcription, and then amplification of the product of this reverse transcription, using a pair of primers specific for each target RNA, from the biological sample, and from a range of synthetic target RNAs comprising the sequence amplified by the pair of d specific primers, (i) (ii) reverse transcription of one or more RNAs of household genes to be quantified, followed by amplification of the product of this reverse transcription, using a specific pair of primers of each household gene RNA, from the biological sample, and from a synthetic household gene RNA range comprising the sequence amplified by the specific primer pair, (iii) time detection of the product of the amplification reactions of step (i) and (ii), (iv) deducing, on the one hand, the amount of RNA or target RNAs in the sample and, on the other hand, the amount of RNA or RNAs of household genes in the sample, and (v) Normalization of the amount of RNA or RNA targets relative to the amount of RNA or RNA from household genes In the process above, the pair of primers for each of the RNAs to be quantified is preferably chosen so as to meet the following criteria: the primers are as close as possible but do not overlap. They allow the amplification of a fragment of size less than or equal to 200 base pairs, preferably less than or equal to 150 base pairs, more preferably less than or equal to 100 base pairs, the nucleotide content G and C primers is between 20 and 80%, it is preferable to avoid nucleotide repetition. In particular, the primers do not contain a repetition of more than three consecutive guanines, the primer fusion temperature is between 58 and 60 ° C., using the Primer Express software as a reference for the calculation of the melting temperatures; the 5 nucleotides at the 3 'end of the primers should not have more than 2 bases G and / or C. The methods of the invention can be used to quantify any type of RNA in a sample, whether it is viral RNA, 16S RNA or messenger RNA coding for viral, bacterial, plant proteins, etc. The term "determined RNA" means an RNA the sequence of which is sufficiently well known to make it possible to design primers specific for this RNA, in particular primers that comply with the criteria set out above.

  In a particular embodiment, the method of the invention makes it possible to quantify the messenger RNAs of one or more cytokines in a biological sample, and comprises the following steps: (i) for each of the messenger RNAs to be quantified, reverse transcription , then amplifying the product of this reverse transcription, using a specific pair of primers of said messenger RNA, from the biological sample, and from a range of synthetic RNA comprising the amplified sequence by the specific primer pair, (ii) real-time product detection of the amplification reactions of step (i), and (iii) deduction of the amount of messenger RNA from each of the cytokines in the sample.

  In order to obtain representative results of the number of cells from which RT-PCR-derived RNA extraction has been performed, step (i) may also involve transcription of the biological sample. reverse, then the amplification of the product of this reverse transcription, with the aid of a specific pair of primers of one or more messenger RNAs of household genes.

  The choice of such a household gene requires very precise validation and it is preferable to combine several household genes to obtain reliable absolute quantification.

  Thus, the method of the invention allows the absolute quantification of the messenger RNAs of one or more cytokines in a biological sample, comprising the following steps: (i) for each of the messenger RNAs of cytokines to be quantified, reverse transcription, then amplification of the product of this reverse transcription, using a specific primer pair of each messenger RNA, from the biological sample, and from a range of synthetic target RNAs comprising the amplified sequence by the specific pair of primers, (ii) the reverse transcription of one or more RNAs of household genes to be quantified, and then the amplification of the product of this reverse transcription, using a specific pair of primers of each household gene RNA, from the biological sample, and from a range of synthetic household gene RNA comprising the sequence amplified by the specific primer pair, (iii) time-sensitive detection. of the product of the amplification reactions of step (i) and (ii) (iv) deduction, on the one hand, of the amount of RNA or messenger RNAs of cytokines in the sample and on the other hand the amount of RNA or RNAs of household genes in the sample, and (v) Normalization of the amount of RNA or messenger RNAs of cytokines relative to the amount of RNA or gene RNAs cleaning.

  In the experiments described below, R2-microglobulin (f12-30 MG) was chosen for work on blood cells, and the expression of this gene is quantified by the same method as that of the gene. target. The results are thus expressed as the number of mRNA copies of the target gene for 1 million copies of f32-microglobulin mRNA.

  The inventors have also developed the genes for ubiquitin C (UBC) and YWHAZ, the expression of which is described as being very stable (Vandesompele et al.). Other household genes can obviously be used in the context of the invention.

  Alternatively, and always in order to quantify the number of cells from which the reaction was made, it is possible to count the cells before extraction of the RNA. However, this is not always possible and often imprecise, and there may also be bias in the extraction of RNA, all the RNA of the counted cells not necessarily being extracted.

  Another possibility would be to express the results as a function of the amount of total retro-transcribed RNA. However, for the same number of cells, this amount may vary according to the phase of the cell cycle where the cells are and depending on the activation state of the cells, which poses a problem for the comparison between a patient and a healthy control that certainly do not have the same status of cellular activation.

  In the method of the invention, the amplification of the reverse transcription product can be carried out by any nucleic acid amplification technique used in molecular biology, such as PCR (polymerase chain reaction), TMA (mediated amplification transcription), NASBA (nucleic acid sequence based amplification), 3SR (self-sustained sequence replication), strand displacement amplification (SDA) and LCR (ligase chain reaction). The PCR will nevertheless preferably be used for this amplification.

  The quantification of different RNAs can be performed in different wells or wells. In the case of a quantification of several RNAs in the same well, the fluorescent intercalent will be replaced by markers allowing distinct readings of each amplification. This can be done using multiplex type techniques.

  The use of a range of reference RNA, allowing to take into account the efficiency of the RT phase, therefore allows an absolute quantification of the messenger RNA considered initially present in the biological sample.

  In a preferred implementation of the method according to the invention, a range of synthetic RNA is therefore constituted for each messenger RNA to be quantified. This range consists, for example, of a series of dilutions of an RNA transcribed from a DNA cassette comprising the phage T7 RNA polymerase promoter 5 'and 3' of the sequence of the amplicon amplified from the messenger RNA, by the specific pair of primers of said messenger RNA. This DNA cassette may be further flanked by endonuclease restriction sites, e.g. EcoRI restriction sites. This can make it possible to clone the constructs obtained in plasmid vectors with which it is possible to transform bacteria, in order to ensure the maintenance and the preservation of these constructions in the long term.

  Another object of the present invention is to allow easy automation of quantification of cytokines of interest. For this purpose, the inventors have developed pairs of primers which make it possible to implement the above method under identical conditions for all the messenger RNAs to be quantified. A process according to the invention, in which the reverse transcription and amplification step is carried out by RT-PCR, and in which all the RT-PCR reactions are carried out under the same temperature conditions, is therefore an integral part of the present invention. This allows in particular the detection of several cytokines on the same plate and the absolute comparison of RNAs encoding several cytokines.

  As mentioned above, the use of fluorescent probes to label the amplicon has the dual disadvantage of being expensive, and to risk introducing a possible bias in the quantification, if the primers used are not strictly specific to the amplicon. Messenger RNA to quantify. In the methods according to the invention, the real-time detection of the amplification product will therefore preferably be carried out by measuring the incorporation of a fluorescent intercalant, for example SYBR Green ITM. Of course, any other fluorescent intercalator, such as BET, Hoeschst 33258, SYBR Gold, YOYO-1, or PicoGreen can be used for this.

  The process developed does not exclude the use of probes or fluorescent primers or any other detection technique for the strict quantification of the amplification product. The detection of the PCR product can indeed be carried out by means of any signal that can be transcribed electronically. Oligonucleotide probes comprising other markers than SYBR Green I are, for example, described in the patents of Kayem et al. US 6,479,340 and US 6,495,323.

  The inventors have developed a set of primers that can be used under standard conditions programmed in a thermocycler, and in a preferred implementation of the methods according to the invention, the RT-PCR reactions are carried out under the following thermocycling conditions:

reverse transcription:

  incubation of the primers: 8 to 12 minutes between 23 and 27 C, preferably 10 minutes at 25 C; reverse transcriptase action: 20 to 80 minutes at 45 to 51 ° C., preferably 30 minutes at 48 ° C. inactivation of the reverse transcriptase: 4 to 10 minutes at 90 to 98 ° C, preferably 5 minutes at 95 ° C .; 2 to 7 minutes at 45 to 55 ° C., preferably 5 minutes at 50 ° C., then 8 to 12 minutes at 90 ° to 98 ° C., preferably 10 minutes at 95 ° C. and then 30 to 45 cycles, preferably 40 cycles, comprising: 12 to 20 seconds between 90 and 98c, preferably 15 seconds at 95 ° C., and 50 to 80 seconds between 55 and 65 ° C., preferably 1 minute at 60 C.

  Specific thermocycling conditions are the following:

- reverse transcription:

  incubation of the primers: 10 minutes at 25 ° C .; reverse transcriptase action: 30 minutes at 48 ° C., inactivation of the reverse transcriptase: 5 minutes at 95 ° C. 15 - 5 minutes at 50 C, then 10 minutes at 95 C; then 40 cycles, comprising: - 15 seconds at 95 ° C., and - 1 minute at 60 ° C.

  Of course, the conditions described above are essentially indicative and in no way restrictive.

  The RT-PCR reactions can also advantageously be carried out in a single step (one step RT-PCR).

  In the examples described hereinafter, all PCR reactions were performed using the Applied Biosystems ™ SYBR Green PCR master mix, on an ABI Prism ™ 7700 machine, under the default thermocycling conditions of: 2 minutes at 50 ° C., 10 minutes at 95 ° C., then 40 cycles of {15 seconds at 95 ° C. and 1 minute at 60 ° C.}.

  The inventors have developed several pairs of primers for human cytokines, as well as 3 housekeeping genes, so that each pair of primers has the following characteristics: specificity: the pairs of primers must be strictly specific to Messenger RNAs to quantify, which means that they do not have to amplify genomic DNA or pseudo-genes. To avoid the amplification of genomic DNA, one technique is to choose at least one primer at the junction between two exons. The choice of primers in different exons, separated by an intron of a length such as the amplification of the genomic DNA would require a duration greater than the duration of the elongation, also makes it possible to avoid the amplification of the Cellular DNA. To avoid amplification of pseudo-genes, case-by-case debugging was necessary. It should be noted that a pair of primers may be specific for a messenger RNA even if one of the primers of this pair (or both) also hybridizes with the cellular DNA and / or with pseudo -Genoa. Indeed, the specificity of a couple is linked to the combination of the two primers. As soon as one of the two primers is strictly specific for the mRNA, or the two primers do not hybridize to the cellular DNA or to a pseudo-gene under conditions suitable for allowing the exponential amplification which characterizes the PCR, a pair of primers will be specific to the mRNA.

  conditions of use: as mentioned above, the pairs of primers have been generated so as to all be usable under the same conditions, and particularly under the same conditions of thermocycling. This implies in particular that they have a melting temperature (Tm) of the order of 58-60 ° C., and that they allow the amplification of a sequence of a size of between 50 and 200 nucleotides, preferably between 75 and 150 nucleotides.

  Example 2 below describes how the primers were developed. The primer pairs are described in Table 1 below: Gene Direct Primer Number (5 '-> 3') SEQ ID Reverse Primer (5 '-> 3') SEQ ID Accession Size No No IL Amplicon -1B XM 010760 CCTGTCCTGCGCTGTTGAAAGA GGGAACTGGGCAGACTCAAA 1 2149 IL-10 AY029171 GCTGGAGGACTTTAAGGGTTACCT CTTGATGTCTGGGTCTTGGTTCT 3 4 108 IL-12p40 AF180563 CATGGTGGATGCCGTTCA 5 ACCTCCACCTGCCGAGAAT 6131 IL-13 NM 002188 ACAGCCCTCAGGGAGCTCAT 7 TCAGGTTGATGCTCCATACCAT August 96 IL-15 U14407 CCATCCAGTGCTACTTGTGTTTACTT CCAGTTGGCTTCTGTTTTAGGAA 9 10 115 IL-18 E17135 CCTGTCCTGCGCTGTTGAAAGA 11 GGGAACTGGGCAGACTCAAA 12 105 IFN-g NM 000619 GTTTTGGGTTCTCTTGGCTGTTA 13 AAAAGAGTTCCATTATCCGCTACATC 14 112 MIF M25639 GCCCGGACAGGGTCTACA 15 CTTAGGCGAAGGTGGAGTTGTT 16 77 TGF-1131 NM 000660 CAAGGGCTACCATGCCAACT 17 AGGGCCAGGACCTTGCTG 18 83 TNF-? XM 041847 CCCCAGGGACCTCTCTCTAATC 19 GGTTTGCTACAACATGGGCTACA 20 97 IL-4 NM 000589 AACAGCCTCACAGAGCAGAAGAC 21 GCCCTGCAGAAGGTTTCCTT 22 101 I2-MG NM 004048 AATTGAAAAAGTGGAGCATTCAGA 23 GGCTGTGACAA AGTCACATGGTT 24 134 YWHAZ NM 003406 ACTTTTGGTACATTGTGGCTTCAA 25 CCGCCAGGACAAACCAGTAT 26 93 Ubiquitin C M26880 ATTTGGGTCGCGGTTCTTG 27 TGCCTTGACATTCTCGATGGT 28 132

Table 1

  In an alternative embodiment of the methods of the invention, for the quantification of messenger RNAs of human cytokines, at least two of the pairs of primers are chosen so that the sequence of primers hybridizing with the messenger RNA considered at least 85% or 90% identity with primer pairs listed in Table 1, considered as reference pairs for each mRNA considered.

  We will consider here, for a pair of primers, that the sequence hybridizing with the messenger RNA considered has at least 85% identity with a given pair if, for each of the primers, the portion of this primer hybridizing with said RNA messenger has at least 85% identity with one of the two primers of the reference torque described in the present application for said messenger RNA. Of course, an identity of 90% is calculated according to the same principle. Several programs exist to calculate the percentage identity between two sequences, among which is the BLASTTM software, which can be used here. In particular, version 2. 06 can be used, as well as the standard version of nucleotide alignment available on the NCBI site or, for sequences between 7 and 20 nucleotides, the specific version for short sequences, available on this same site (www.ncbi.nlm.nih.gov/BLAST/). The similar sequences thus envisaged may be of the same length as the sequences explicitly described in the present text, or of a different length, then preferably comprised between 15 and 35 bases. In particular, primers differing from primers described by a modification at their 5 'end (for example an addition of nucleotides that are not complementary to the messenger RNA sequence), but which retain the characteristics of specificity and conditions of use described herein. above, can be easily used instead of specific primers developed by the inventors. By abuse of language, and for greater clarity of the present text, it will be said that a pair of primers whose sequence hybridizing with the messenger RNA considered has at least 85% identity with a couple described herein. request, that it has at least 85% identity with said pair of primers, even if this identity is only calculated for the part of each primer that is likely to hybridize with the messenger RNA.

  In the methods of the invention, it is also possible to control the efficiency of the RNA extraction phase from the biological sample. For this, a known amount of heterologous RNA is introduced into the sample before extraction. By "heterologous RNA" is meant herein any RNA which comprises an amplifiable sequence by RT-PCR with primers which do not allow the amplification of an identical sequence from the biological sample. It may be an RNA from a different species, for example bacteria or yeast when the sample comes from an animal, or a synthetic RNA. This heterologous RNA is then quantified after RT-PCR (or equivalent reaction), which makes it possible to control the extraction phase.

  Also in order to control the extraction yield, it is possible to introduce into the biological sample to be analyzed a known number of heterologous cells. The level of expression of the RNA of a household gene (or other) of these cells is then quantified after extraction. Of course, this gene is chosen such that it comprises an amplifiable sequence by RT-PCR with primers which do not allow the amplification of an identical sequence from the biological sample. The ratio between the number of messenger RNA copies of this heterologous cell gene and the number of heterologous cells introduced into the sample makes it possible to evaluate the yield of the extraction.

  The invention also relates to a set of nucleotide primer pairs intended in particular for use in the methods of the invention, for quantifying messenger RNAs of one or more cytokines in a biological sample, and in which at least two pairs primers, preferably three, are selected from, or have an identity level of at least 85% or 90% with the primer pairs of Table 1.

  Another subject of the invention is a kit for detecting disorders related to the regulation of Th1 / Th2 lymphocytes, comprising a set of primer pairs as described above, characterized in that the primer pairs are specific. messenger RNAs of cytokines selected from IL-10, IL-13, TGF-I3, TNF-α, and IFN-γ. If desired, such a kit may also contain one or more specific cytokine messenger RNA primer pairs selected from IL-2, IL-4, IL-5, and TNF-f3.

  The present invention also relates to a kit for detecting inflammatory disorders or innate immunity disorders, comprising a set of primer pairs as described above, in which the primer pairs are specific for messenger RNAs. of cytokines selected from IFN-γ, TNF-α, TGF-f3, IL-1-f3, IL-12β35, IL-12β40, IL-15 and IL-12. 18. Such a kit may also further comprise one or more specific cytokine messenger RNA primer pairs selected from IFN-α, IL-6, IL-8, IL-21, IFN-j3, INOS and IL-16.

  Two other kits, intended to quantify the messenger RNAs of cytokines connected to IL-10, or connected to IL-12, are also part of the invention. The first comprises a set of primer pairs as described above, in which a pair of primers is specific for the messenger RNA of IL-10, one or more pairs of primers are specific for selected from the human E32-microglobulin genes, ubiquitin C and the YWMAZ gene, and other primer pairs are specific for messenger RNAs of cytokines selected from IL-19, IL-20, IL-22 and IL-24. The second one, dedicated to IL-12-related cytokines, comprises a set of primer pairs as described above, in which a pair of primers is specific for the messenger RNA of IL12p40, one or several pairs of primers are specific for messenger RNAs of household genes selected from the genes of human 132-microglobulin, ubiquitin C and the YWMAZ gene, and other pairs of primers are specific for messenger RNAs of cytokines selected from IL-12p35, IL-23, IL-27 and AK155.

  One of the purposes of the detection kits according to the present invention is to be used clinically to evaluate the state of the immune system of a patient, without requiring the development of the conditions for performing the RT-PCR for each messenger RNA. sought, with a view to directly obtaining an absolute quantification of messenger RNAs of cytokines involved in the disorders mentioned above.

  This absolute quantification can be achieved through the use of standard RNA ranges. A kit according to the invention, as described above, and further comprising, for each pair of specific primers of a messenger RNA, a synthetic RNA comprising the sequence of said messenger RNA which is amplified by the pair of primers, therefore also part of the present invention.

  The kits of the invention will also advantageously include an explanatory note intended for the user.

  Assuming that the efficiency of the RT phase could be controlled, a DNA range comprising the sequence of the complementary DNA which is amplified by the pair of primers could also be used. Alternatively, the user of the kits according to the present invention can produce a standard RNA range from a DNA template. The invention therefore also relates to a kit as described above, characterized in that it further comprises, for each specific pair of primers of a messenger RNA, a synthetic DNA comprising the sequence of the complementary DNA which is amplified by the pair of primers.

  In the kits of the invention comprising an RNA or a synthetic DNA intended to constitute a reference range, this synthetic nucleic acid can be present either directly in the form of a standard RNA range or in the form of a standard RNA. This is a determined concentration from which the user will make dilutions, either in the form of precipitated and dried RNA, for example freeze-dried.

  The stock solution of the standard range (DNA or RNA) may also be present in the form of nucleic acid in solution in ethanol or isopropanol. The user will then have to centrifuge this solution in order to pellet the nucleic acid before resuspending the pellet in an appropriate buffer (water type + Rnase inhibitors or DEPC treated water), and to determine by reading the optical density the concentration of the solution obtained. This will then be brought back to the concentration corresponding to the first point of the standard range.

  As mentioned above, the synthetic RNA can be obtained by in vitro transcription from a DNA cassette comprising the phage T7 RNA polymerase promoter 5 'and 3' of the sequence to be amplified.

  Another kit according to the invention may therefore contain a DNA construct serving as a transcription matrix for obtaining the standard RNA and, where appropriate, the reagents necessary for the in vitro transcription of this matrix. As mentioned above, the user will, after transcription and purification, quantify the synthetic RNA in order to generate the standard RNA range. A notice with instructions to that effect may be included in the kit in question.

  The kits according to the invention may also comprise a heterologous RNA or heterologous cells with respect to the biological samples with which they are intended to be used, as well as a pair of primers specific for a sequence included in this RNA or in the RNA from these cells. When using these kits, a quantity of the heterologous RNA or heterologous cells will be mixed with each biological sample to be treated, before the extraction of the RNA, in order to control the efficiency of the extraction phase. , as mentioned above.

  Such a kit therefore preferably comprises primers specific for this heterologous RNA or an RNA sequence of these heterologous cells. A control protocol for this effectiveness can then be described in the explanatory note possibly present in the kit.

  The synthetic DNA can be obtained by PCR using either the primers specific for the RNA under consideration, or a modified version of these primers (addition of restriction sites, etc.). The obtained amplicon can be cloned in a plasmid vector (either using restriction sites or by using cloning kits of PCR products type TA cloning kit of InVitrogen (D).) This not only allows a better conservation of the construction, but also a better determination of the concentrations of standard solutions, because for an identical number of amplicons, the optical density value will be higher, since it will take into account the nucleotides making up the vector.

  A kit according to the invention may also comprise a data processing software capable of compiling the results obtained from the standard ranges, the household genes, and the biological sample, and directly giving the amount of messenger RNA. of each cytokine tested in the biological sample. This software can be carried by any media, including a floppy disk or a card directly implantable on the thermal cycler used. If necessary, it can be reduced to a macro in Microsoft Excel or other spreadsheets, to automate the main steps of data processing.

  According to a preferred embodiment of sets of primer pairs, or kits of the invention, these sets or kits comprise specific primer pairs of messenger RNAs of at least three, four, five, six, seven, eight, nine, ten, or eleven cytokines.

  In another aspect of the invention, the sets of primer pairs or kits described above preferably comprise primer pairs specific for the messenger RNAs of at least one, two, three or four housekeeping genes. These housekeeping genes may for example be chosen from the genes of [3-actin (ACTB), p-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPD), hydroxymethylbicyclic synthase (HMBS), hypoxanthine phosphoribosyl transferase 1 (HPRT 1), ribosomal protein L13a (RPLI3A), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TBP), ubiquitin C (UBC), and the zeta peptide of the tyrosine-3-monooxygenase I activation protein tryptophan monooxygenase (YWHAZ). Examples of primers that can be used to amplify sequences of these household genes are given in the Vandesompele article, De Preter et al. 2002. The examples and figures presented below without limitation will highlight certain advantages and features of the present invention.

  Figure 1: Diagram of the real-time quantitative RT-PCR protocol using a range of recombinant standard RNAs.

  Figure 2: Construction of a standard RNA range. The specific primer pair generates an amplicon that serves as a template for a second PCR using the specific primers modified by the 5 'fusion of the T7 promoter sequence followed by an EcoRI restriction site. The PCR product is purified before being used as an in vitro transcription matrix. The standard RNA product is purified, quantified to achieve a standard RNA range. This range will undergo RT-PCR and will serve as a reference for the absolute quantification of the number of copies of the mRNA considered.

  Figure 3: Comparison protocol of the RNA versus DNA range approaches.

  Lane I: After reverse transcription, a standard RNA cDNA is serially diluted 10 fold to generate a cDNA range. Lane II: The standard RNA is serially diluted by a factor of 10 and each dot undergoes a RTPCR. Lane III: A sequence of DNA sequence corresponding to that of the standard RNA is amplified by PCR. This route is that conventionally used for the quantification of mRNAs. The real-time PCR conditions are identical for the 3 channels (same PCR mix, same plate). The corresponding amplification efficiencies are calculated from the slopes of the standard lines. The efficiency of reverse transcription is equal to the ratio between EII and El. Efficacy of PCR (E1) Efficacy of RTPCR (EN) Efficacy of PCR (EIII) Figure 4: Validation of primers. The specificity of the pairs of primers used is validated by analysis on agarose gel (A) and by sequencing. The dissociation curves (B) also provide information on the formation of primer dimers. Finally, the selected primers are used in real-time RTPCR to amplify a range of standard RNAs specific for said pair of primers (example of the (32-pglobulin: C).

  Figure 5: Dissociation curves of human amplification products for 132-microglobulin, MIF, IFN-γ, IL-1β3, IL-15, IL-12p40, IL-10, TNF, IL-13, IL-18 and TGF- (3-1) After 40 amplification cycles, the amplicons are subjected to a gradual increase in temperature from 60 to 85 C. A sudden decrease in Fluorescence accompanies the dissociation of the two DNA strands of the amplification product when they reach their melting temperature.The specificity of the primers used is validated by the presence of a single peak.

  Figure 6: Dissociation curves of human amplification products for ubiquitin C (A.) and YWHAZ (B.). The specificity of the primers used is validated by the presence of a single peak.

  Figure 7: Dissociation curves of the simian amplification products for R2-microglobulin, MIF, IL-12p40, IL-10, IL-15 (A) and IFN-γ, the GAPDH and TNF-a (B.). The specificity of the primers used is validated by the presence of a single peak.

  Figure 8: Determination of optimal primer concentrations for TNF-a. The sense and antisense primers were used at 50, 300 or 900 nM final to amplify TNF-α transcripts by RT-PCR in real time.

  Figure 9: Determination of the sensitivity of the technique. Total RNA from a decreasing number of cells was subjected to RT-PCR for the f12-microglobulin gene. In parallel, a standard RNA range of this same gene has undergone the same steps. Thus, it has been determined that the technique of the present invention permits the extraction and amplification of RNA from at least 10 cells. The improvement in the yield of the RNA extraction phase is underway and should make it possible to increase this sensitivity.

  Figure 10: Sensitivity of RNA ranges. A standard RNA copy number range of each transcript was retro-transcribed and then amplified by real-time PCR to determine the minimum number of amplifiable copies. For IL-1 f3 transcripts, our technique allows the detection of 10 copies of mRNA.

  Figure 11: Intra-experimental reproducibility of the technique for the f32-microglobulin and IL-1 f3 genes. Four identical ranges of standard RNA were retro-transcribed and PCR amplified. The panels A. and B. show the average of the threshold cycles and the difference to this average for each of the points. One of the ranges served as a basis for determining the number of copies present in the other 3 ranges. Charts C. and D. represent the average of these calculated values and the standard deviation at this average for each of the points as a function of the number of theoretical copies.

  Figure 12: Intra-experimental reproducibility of the technique for the IL-15 and IL-18 genes. Four identical ranges of standard RNA were retro-transcribed and PCR amplified. The panels A. and B. show the average of the threshold cycles and the difference to this average for each of the points. One of the ranges served as a basis for determining the number of copies present in the other 3 ranges.

  Charts C. and D. represent the average of these calculated values and the standard deviation at this average for each of the points as a function of the number of theoretical copies.

  Figure 13: Intra-experimental reproducibility of the technique for IFN-γ and TGF-R1 genes. Four identical ranges of standard RNA were retro-transcribed and PCR amplified. The panels A. and B. show the average of the threshold cycles and the difference to this average for each of the points. One of the ranges served as a basis for determining the number of copies present in the other 3 ranges.

  Charts C. and D. represent the average of these calculated values and the standard deviation at this average for each of the points as a function of the number of theoretical copies.

  Figure 14: Intra-experimental reproducibility of the technique for the genes of ubiquitin C and YWHAZ. Four identical ranges of standard RNA were retro-transcribed and PCR amplified. The panels A. and B. show the average of the threshold cycles and the difference to this average for each of the points. One of the ranges served as a basis for determining the number of copies present in the other 3 ranges. Charts C. and D. represent the average of these calculated values and the standard deviation at this average for each of the points as a function of the number of theoretical copies.

  Figure 15: Inter-experimental reproducibility of the technique for the R2-microglobulin and TGF-R1 genes. Eight different ranges of standard RNA were retro-transcribed and PCR amplified on different days. The panels A. and B. show the average of the threshold cycles and the difference to this average for each of the range points. Charts C. and D. represent the average of the calculated values and the standard deviation at this average for each of the points as a function of the number of theoretical copies.

  Figure 16: Comparison of the RT-PCR efficiencies between a standard range and a cell sample. A range of standard RNA for the $ 10 to $ 10microglobulin copy was amplified by RT-PCR. The slope of the resulting standard line (A.) is equal to -3.305. This slope is very close to that resulting from the standard line B. corresponding to a cell density range ranging from 10 to 105 cells whose total RNA has undergone the same RT-PCR conditions as the standard range for the amplification of MRNA of (32-microglobulin (= 3.307).

  Figure 17: Comparison of the efficiencies and sensitivities between the method of the invention and the TagManTM kit for the amplification of transcripts of TNF-a. A range of total cellular RNA was retrotranscribed and then amplified by real-time PCR using either the method of the invention or the TagMan ™ commercial kit. The standard lines obtained make it possible to determine the efficiency and the sensitivities of the two techniques. Under the conditions tested, the method of the invention is as sensitive and more effective than TaqMan TM considered as a reference technique.

  Figure 18: Kinetics of transcription and secretion of MIF (A.) and TNF-α (B) A. Kinetics of transcription and secretion of MIF by peripheral blood mononuclear cells from a healthy donor after 3, 6 and 9 hours of culture in the presence of LPS (10 ng / mL), SAC (0, 0075%), red blood cells parasitized by Plasmodium falciparum trophozoites stage (20: 1) or healthy red blood cells (20: 1) ). As this cytokine is partly preformed, there is no direct relationship between transcript levels and secreted protein levels. Secretion of MIF is measured in culture supernatants by ELISA (R & DTM) B. Kinetics of transcription and secretion of TNF-α by mononuclear peripheral blood cells from a healthy donor after 3, 9 and 18 hours of culture in the presence of LPS (10 ng / mL), SAC (0.0075%), red blood cells infected with Plasmodium falciparum at the trophozoite stage (20: 1) or healthy red blood cells (20: 1). Since this cytokine is not stored in the cell, transcription of the mRNA precedes by a few hours the secretion measured in the culture supernatants by ELISA (BioSource). There is therefore correlation between the transcriptional activity and the secretion of the corresponding protein.

  Figure 19: Gene transcription of different cytokines by monocytes isolated from healthy donors. Isolated monocytes were incubated for 0.5, 1, 2, 3, 6 or 9 hours in the presence of LPS (100 ng / mL) or SAC (0.0075%). Cytokine transcripts are quantified and standardized for one million copies of 62-pglobulin RNA. The histograms represent the ratio of the number of RNA target copies to the R2-microglobulin mRNA (average of 2 donors).

  Figure 20: Transcriptional capacity of TGF-f.1 in control subjects or patients with malaria.

  PBMCs of control individuals (Asymptomatic Controls) or patients suffering from malaria (Malaria patients) were removed and stimulated for 22 hours by LPS (100 ng / mL). The total RNA of these PBMCs was extracted to quantify the number of TGF-R1 transcripts. The histograms represent the copy number of TGF-R1 mRNA for one million copies of R2-microglobulin mRNA. *: statistically significant difference (p <0.05) 2874026 26 Figure 21: Development of conditions for quantitation of IL-4 mRNA. The specificity of the chosen primers is validated by the presence of a single peak on the dissociation curve (A.) and by sequencing of the product. A standard RNA concentration range is amplified by RT-PCR to validate its use for quantification of IL-4 (B.) mRNAs. This is performed on PBMCs of 2 healthy donors (D16 and D18) stimulated with LPS, SAC or red blood cells parasitized by Plasmodium falciparum for 0.5, 1, 2, 3, 6 or 9 hours (C.).

EXAMPLES

  For all of the following examples, the following materials and methods were used: Cell Stimulation Human mononuclear peripheral blood cells (PBMCs) from healthy donors were isolated using Ficoll ™ 1.077 (Nycomed ™) One Million Living Cells were distributed in 48-well flat bottom culture plates (Costar ™), and stimulated with LPS (10 ng / ml, E. coli 0111: B7, Sigma ™), PHA-L (10 μg / ml, Sigma ™). , SAC (0.0075%, Pansorbin CellsTM), live P. falciparum schizonts (1% final parasitaemia, strain FCR3) or intact and uninfected red blood cells.

  The cells were harvested for RNA extraction after 3, 6, 9, 12, 18, 24 and 48 hours of stimulation, then resuspended in RNA PLUS buffer (Q-Biogene ™, 500 μl for 10 6 cells). .

  RNA extraction and reverse transcription

  Total cellular RNA was extracted using the RNA-PLUS kit from Q-Biogene, following the supplier's instructions and resuspended in RNase-free water (AmbionTM). The amount of total RNA was determined by optical density at 260 nm. Complementary DNA was obtained using the "TagManTM Reverse Transcription Reagents" reagents (Applied Biosystems ™) in a 50 μl reaction volume.

  Amplification of the Complementary DNA A real time quantitative polymerase chain reaction (PCR) was carried out on an AB1 Prism 7700TM thermocycler using the SYBR-Green PCR master mixTM buffer (Applied BiosystemsTM). ) in a final volume of 30 pI. The thermocycling conditions were the default conditions: 50 C for 2 minutes, 95 C for 10 minutes and 40 cycles of {95 C for 15 seconds and then 60 C for 1 minute}. Each point has been duplicated. The primers used for each amplification (each at 900 nM final) are described in Table 1.

  Construction of External RNA Standards Modified primers were used on the cellular cDNA to construct the transcription matrix. These primers were the primers specific for the genes considered, fused at their 5 'end to the T7 promoter sequence. Each matrix was then purified on a silica column (NucleospinTM, Macherey-NagelTM) before being transcribed in vitro using the MegaShort Script Transcription KitTM kit (AmbionTM) The concentrations of the standard RNAs were calculated by optical density at 260 nm. Serial dilutions were performed for each external standard, in a range from 10 copies per μL to 1012 copies per μL, in RNase free water. These dilutions were aliquoted and stored at -80 ° C. These standards then underwent the same reverse transcription as cellular RNA.

  Quantitative Analysis External standard curves and quantification were obtained using the SDS 1.9 software. From these standard curves, the initial amount of target messenger RNAs was determined for each of the samples, and standardized for the microglobulin.The results are expressed as the number of copies of messenger RNA per million copies of messenger RNA. (32-microglobulin.

  ELISA test The ELISA tests were performed according to the supplier's instructions (TNF-alpha: BioSource and MIF: R & D).

  Example 1: Standard RNA Curve 1.1: Generation of Standard Curve The nested PCR technique (Nested-PCR) was used to flank each specific amplicon of a T7 polymerase promoter sequence. This construct was used to generate a standard RNA that was loaded onto a gel and migrated to verify its clonality (results not shown). The molecular weight of each standard was calculated on the basis of its expected sequence, and then solutions ranging from 101 to 1012 standard RNA copies per microliter were made.

  These serially diluted solutions were subjected to reverse transcription, and the DNA was amplified by doubling each point, to generate a standard curve, by pointing the threshold cycle (CT) against the logarithmic value of the number of copies. Starting RNA for each dilution. Figure 2 shows an example of these curves for IFN-γ. Each generated curve showed an RT-PCR efficiency greater than 84% and a dynamic range of at least 7 orders of magnitude. This allowed reliable and reproducible quantification of mRNA from a cell sample.

  1.2: Comparison between standard RNA and DNA curves In order to compare the results generated by the use of a standard RNA range with those resulting from the use of a standard DNA range, the following experiment was performed. 10 15

  The DNA templates for transcription of the standard IL-1 1, IFN-γ and TNF-α RNAs were purified and quantified by OD in order to achieve concentration ranges of 10 to 10 μM. copies at 1012 copies / pL.

  There were therefore two types of ranges in which the two constructions have almost the same sequence.

  The standard RNA ranges were subjected to a reverse transcription step and 5 μL of the RT product was amplified by real-time PCR.

  In parallel, a standard RNA sample was retro-transcribed, and the corresponding cDNA was serially diluted by a factor of ten. A range of cDNA has thus been generated and subjected to amplification (same mix, same final volume as the RNA and DNA ranges).

  The protocol is shown schematically in Figure 3.

  The standard straight lines resulting from these three ranges have the characteristics indicated in Table 1 below: According to the PCR theory, the number of copies between two cycles increases by a factor X which is equal to 2 if the PCR is 100% effective.

  The E efficiency of a PCR is given by E = X-1, or X = 10-1 / slope. Thus, the efficiencies indicated below are given.

  Standard RNA CHANNEL III: Standard DNA Route I: PCR Route II: RT-PCR slope efficiency Efficacy of RT (11/1) Slope efficiency Slope efficacy IL-1 [3 -3,1966 1,05 -3,4794 0,94 -3, 067 1.11 0.84 IFN-y-3.161 1.09 -3.843 0.82 -2.944 1.19 0.76 TNF-a-3.556 0.91 -3.61 0.89 -3.503 0.93 0.97 Table 2: Influence of RNA range versus DNA range approaches on the quantification of transcripts. The slope and efficiency data for the three types of range are given. The reverse transcription (RT) efficiencies are calculated by the ratio between the efficiency of RT-PCR (lane II) and that of PCR (lane I).

  It thus appears that the efficacies of the RT-PCRs are lower than those of the PCRs for the 3 genes.

  This is due to the RT phase since the efficiencies of PCR on cDNA and DNA are similar for each gene.

  The efficiencies of cDNA PCRs being similar to those of DNA PCRs, the difference in efficacy between RT-PCR and PCR is attributable to the RT phase which is not 100% effective, unlike advanced by most authors to justify the use of a DNA array for mRNA quantification.

  This is a first bias to take into account to reliably quantify the rate of transcripts. However, if this bias were constant, the comparison between the experiments would be possible.

  Experience shows that the efficiency of the RT phase differs according to the RNAs. The quantification bias provided by the RT phase therefore exists and varies. This can therefore in no way allow the use of DNA ranges for the reliable, reproducible and accurate quantification of messenger RNAs.

  Indeed, if we compare the data obtained based on the standard straight lines from the DNA or RNA ranges to quantify a known number of TNF-α transcripts, the following results are obtained: Nb of copies Nb of copies Nb of real initial copies initial calculated by initial calculated by route II (RNA) route III (DNA) Experiment 1 5.10 '5.2.10' 7.7.105 Experiment 2 5.10 '4.9.10' 5.7.105 Table 3: Influence of RNA versus range approaches DNA on the quantification of transcripts Based on a range of DNA, the number of transcripts present in the sample is therefore greatly underestimated. In addition, the degree of underestimation varies according to the transcripts studied.

  All of these data justify the use of a standard RNA range for the reliable and reproducible quantification of transcripts and demonstrate the lack of accuracy and usability of using a range of DNAs.

  Example 2: Primers Example 2.1: Validation of the primers Primer pairs were drawn from the genomic sequences published using the Primer ExpressTM software (Applied BiosystemsTM), by best respecting the following criteria: - Choosing primers the closer to each other as long as they do not overlap - Keep GC content between 20 and 80% - Avoid nucleotide repetition. This is particularly true for guanine for which a sequence of 4 guanines or more should be avoided.

  - Choose primers with a melting point between 58 and 60 C when using the Primer ExpressTM software The 5 nucleotides at the 3 'end should not contain more than 2 G and / or C bases. The specificity of the primers for the target cDNAs was provided by drawing direct and reverse primers amplifying a product covering an intron and which did not lead to the amplification of pseudo-genes or other related genes. Specificity was estimated by ethidium bromide agarose gel analysis, dissociation curves (Figs 4-7) and sequencing PCR amplification products (results not shown).

  Each amplicon was cloned into a pCR2.1 vector using the lnvitrogen ™ (TA cloning kit ™) kit, and sequenced. The sequences all had at least 98% identity with the expected sequence (results not shown).

  Example 2.2: Optimization of the concentration of the primers It is a question of testing the effect of a variation of the concentration of primers on the amount of RNA copies detected. To do this, the lo sense and antisense primer concentrations ranged from 50 to 900 nM. The concentrations of the primers were optimized to determine the minimum primer concentrations giving the lowest threshold cycle (CT), and therefore the highest sensitivity, and the maximum signal-to-noise ratio in fluorescence (ARn). while minimizing nonspecific amplifications (results not shown).

  Figure 8 shows the results obtained for the detection of TNF-a. For this gene as for all others, the optimal concentration pair is 900nM for the sense and antisense primer.

  Example 3: Determination of the sensitivity of the technique: Minimum sample size and minimum number of copies In order to determine the sensitivity of the technique, a range of cell density ranging from 105 cells to 1 cell was performed. The total RNA of these cells was extracted and retro-transcribed. One-tenth of the obtained cDNA was amplified by PCR for the 32-microglobulin gene. In parallel, a standard RNA range of this same gene has undergone the same steps of RTPCR. The results of this experiment are presented in FIG. 9. They show that this technique makes it possible to reliably detect the transcripts of the β-2-microglobulin gene in at least 10 cells.

  It should be noted that this is not an RNA dilution corresponding to a cell equivalent of 10 but rather of RNA extracted directly from 10 cells. If we reason in standard RNA dilution, the sensitivity is 10 copies.

  For each of the genes developed, the sensitivity of the technique in terms of detectable copy number has also been established. This varies according to the cytokines between 10 and 100 copies (FIG. 10). Example 4: Intra- and inter-experimental variations The reproducibility of the technique was studied. Intra-experimental variability was calculated from standard quadruplicate RNA range points for the R2-microglobulin, ubiquitin C, YWHAZ, IL-1G, IL-15, IL-18, IFN-gamma and TGF-M. The average threshold cycles for each point as well as the standard deviations at this average have been calculated and are shown in Figures 11, 12, 13 and 14 (A and B).

  Similarly, one of the ranges served as a standard for calculating standard RNA quantities for the other 3 points. The average of the quantities obtained as well as the standard deviations at this average are shown in FIGS. 11, 12, 13 and 14 (CetD).

  Inter-experimental variability was determined for the R2-microglobulin and TGF-111 genes from 8 independent experiments performed several days apart and using different batches of PCR mix. The same strategy as for intra-experimental variability was used and the results are shown in Figure 15.

  These results show that the technique of the present invention is very reproducible, both at the intra-experimental level and at the interexperimental level. This is a major asset for the reliability and comparison of the results obtained for different samples processed at different times.

  Example 5 Comparison of the Efficacies of RT-PCR Between the Range and the Sample As in Example 4, the efficiency of RT-PCR varies from one gene to another and according to the experiments for the same gene.

  It is therefore possible that this variation causes a bias in the quantification if it occurs between the range and the samples.

  The RT-PCR efficiencies between a range of 132-microglobulin and extracted cell RNAs of 10, 100, 1000, 10,000 or 100,000 cells were therefore compared.

  Thus, the standard and cellular RNAs each comprise an RNA concentration range whose points are spaced by a factor of 10.

  These two populations of RNA were simultaneously subjected to the same RT-PCR conditions for the amplification of 132-15 microglobulin mRNAs.

  The results are shown in Figure 16.

  They show that the slopes of the standard straight lines are almost identical and that therefore the RT-PCR efficiencies are very close between the standard range and the cell sample (100.7% and 100.6% respectively).

  Thus, the variability of the efficiency of RT-PCR does not introduce bias in the quantification of mRNAs under the conditions used in this experiment. The results obtained by this technique are therefore reliable.

  Example 6 Comparison of the Efficiencies of RT-PCR Between the Method of the Invention and the TagMan TM Kit The method of the invention was compared with the Tagman ™ technique, a reference technique in this field, for the TNF-α gene. . Monocytes were stimulated for 6 hours with LPS. Total RNA was extracted from 106 cells and serially diluted. This range of total cellular RNA was amplified for TNF-α gene mRNA, in parallel, on the same plate and under the same thermocycling conditions using either the method of the invention or TagmanTM chemistry (Applied- Biosystems).

  The results of this test are shown in FIG. 7. At the dilutions studied, it appears that the method of the invention is as sensitive as the TagmanTM reference technique. In addition, the present method shows a slightly higher efficiency than the TagmanTM technique: 105% versus 80% respectively corresponding to slopes of -3.2 (technique 10 of the invention) and -3.9 (TagmanTM technique) Example 7: Validation of internal standards (housekeeping genes) The choice of an endogenous gene stably expressed and which can be used as an internal standard (housekeeping gene) is a prerequisite for accurate RT-PCR quantification . The internal standard is chosen based on the clinical evaluation of the patient and the tissue origin of the biological sample. Since cytokine analysis requires an internal standard that can be used on peripheral blood leukocytes, 3 known genes for the stability of their expression in leukocytes have been studied: R2-microglobulin (P2-MG), ubiquitin C (UBC) and the YWHAZ gene. The stability of the amount of transcripts of these 3 genes was compared by the method of the invention under different activation conditions.

  PBMCs from healthy donors were stimulated for 3 hours with PBS or PHA (phytohaemaglutinin) and then separated into two identical aliquots. The total cellular RNAs were extracted at two days' intervals then the same volume of RNA was retro-transcribed, and the transcripts of the 3 genes amplified by RT-PCR, under the same conditions. Table 4 below shows the results obtained: average obtained and in parentheses the results of the two extractions. These data show that microglobulin gives reproducible results, with less than 12% variability between the different experiments, whereas the results for the ubiquitin C and YWHAZ genes show a variability of up to 47%. Β-microglobulin was therefore chosen as the internal standard for future absolute quantification experiments.

  TO (non-stimulated) LPS 3h PHA 3h 1.24.10 '1.48.10' (1.00-107-1.49.10 ') (1.17.107-1.79.10') 2.04.105 2.39.105 (0.62.105-3) , 46.105) (1.51.105-3.28.105) 1.89.105 2.80.105 (0.78.105-3.01.105) (2.25.105-3.34.105) Table 4: Choice of internal standard. For each gene tested, appear on the first line the average of the two extractions, and on the second line, the individual result of the two extractions. The tests were performed without stimulation (TO) or after 3 hours of stimulation with LPS or PHA.

  Example 8: Kinetics of transcription of M1F and TNF-α factors Figure 18 shows the kinetics of transcription and production of MIF (A) and TNF-a (B) by PBMCs of a healthy donor stimulated by LPS, SAC, Plasmodium falciparum-parasitized red blood cells or healthy red blood cells for 3, 9 and 18 hours.

  The results show an increase in TNF-α transcription at about 3 hours after stimulation, a transcription that precedes the secretion of the TNF-α protein (B.). On the other hand, the MIF gene is strongly transcribed and constitutively in the basal state (TO), and neither stimulation with LPS nor with SAC increases this transcription.

  This demonstrates that the method of the invention is validated on samples stimulated in vitro. In addition, it highlights the complementarity of the RNA and protein assay approach for cytokines (32-MG

UBC

YWHAZ

  1,37,10 '(1,11,10'-1,64,10') 1,98,105 (1,33,105-2,64,105) 3,36,105 (2,60,105-4,13,105) neoformed (TNF-α) compared to preformed cytokines (MIF). Finally, these results confirm the literature data that report that MIF is present in cells both in the form of preformed proteins ready to be secreted, and in the form of mRNAs that can be rapidly

  translated without any transcript.

  Example 9: Kinetics of Transcription of the Genes of Different Cytokines In vitro stimulation experiments with LPS and SAC were carried out on the monocytic and lymphocyte subpopulations, isolated from the blood, for 0.5, 1, 2, 3 , 6 and 9 hours. The extraction of the corresponding RNAs was carried out and the quantification of the messenger RNAs coding for the IL-1 cytokines (3, IL-15, IL-4, IL-18, IL-10, MIF, IL12p40, TGF-RI, IL -13 and TNF-a was performed and related to the amount of mRNA coding for beta-2 microglobulin, chosen as a household gene (internal standard).

  Figure 19 shows the result obtained with the purified monocytes of one of the donors. No significant increase in the amount of IL-15, IL-18, MIF, or TGF- (1) mRNA was detected, and for both IL1 G and IL-10 both stimulants induce comparable kinetics of transcript accumulation, whereas an accumulation of early IL-4 and IL-13 transcripts in response to LPS (2 hours) not found for SAC As for IL-12p40, there is an earlier accumulation of transcripts by stimulation with LPS than with SAC Finally, the accumulation of transcripts TNF-a is more prolonged in time after stimulation with the SAC, compared to stimulation with LPS.

  Example 10: Transcription capacity of TGF / 1

  RNAs from peripheral blood mononuclear cells from malaria patients or control individuals, after stimulation with LPS, were extracted. The quantification of the mRNAs of the genes for 132 microglobulin and TGF-131 was performed. Figure 20 shows the difference in transcriptional capacity of TGF- (31 between patients with malaria and control individuals.

  The cells of malaria patients show higher TGF-81 transcription than control subjects after PBS stimulation (p = 0.036, statistically significant difference *).

  EXAMPLE 9l Kinetic Quantification of IL-4 IARNm Primer for IL-4 were drawn and IL-4 amplification conditions validated. Figure 21 shows the dissociation curve of the selected primers (A.), as well as the line showing the efficiency of the amplification (B.). In order to validate this cytokine, messenger RNAs coding for IL-4 from PBMCs derived from 2 healthy donors (donors 16 and 18) were quantified, after stimulation of these PBMCs, between half an hour and 9 hours. with LPS (100 ng / ml), SAC (0.0075%) or Plasmodium falciparum-infected red blood cells at the concentration of 20 parasites for a leukocyte. The expression kinetics of IL-4 appears comparable for both donors (C.)

BIBLIOGRAPHY

  Bustin, S. A. (2000). "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays." J Mol Endocrinol 25 (2): 169-93.

  Ginzinger, D. G. (2002). "Quantitation Gene using real-time quantitative PCR: an emerging technology hits the mainstream." Exp Hematol 30 (6): 503-12.

  Giulietti, A., L. Overbergh, et al. (2001). "An overview of real-time quantitative PCR: applications to quantify cytokine gene expression." Methods 25 (4): 386-401.

  Stordeur, P., L. F. Poulin, et al. (2002). "Cytokine mRNA quantification by real-time PCR." J Immunol Methods 259 (1-2): 55-64.

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Claims (26)

  A method for the absolute quantification of one or more target RNAs in a biological sample, comprising the following steps: (i) for each of the target RNAs to be quantified, reverse transcription, then amplification of the product of this reverse transcription, at the using a specific primer pair of each target RNA from the biological sample, and from a synthetic target RNA range comprising the sequence amplified by the specific primer pair, i0 (ii) the inverse transcription of one or more RNAs of household genes to be quantified, then the amplification of the product of this reverse transcription, using a pair of primers specific for each RNA of household genes, starting from the biological sample, and from a range of synthetic household gene RNA comprising the sequence amplified by the specific primer pair, (iii) real-time product detection of step amplification reactions (i) and (ii), (iv) one-off deduction of the amount of RNA or target RNAs in the sample and secondly the amount of RNA or RNAs of household genes in the sample and (v) Normalization of the amount of target RNA or RNA relative to the amount of RNA or RNAs of household genes.   2. Method according to claim 1, wherein the pair of primers is chosen so as to meet the following criteria: the primers do not overlap and allow the amplification of a fragment of size less than or equal to 200 base pairs , preferably less than or equal to 150 base pairs, more preferably less than or equal to 100 base pairs, the nucleotide G and C content of the primers is between 20 and 80%, the primers do not contain a repetition of more than three consecutive guanines, the melting point of the primers is between 58 and 60 ° C., the 5 nucleotides at the 3 'end of the primers comprise at most 2 bases G and / or C. The method according to claim 1 or 2, wherein the determined RNA is a viral RNA, a 16S RNA or a messenger RNA.   4. Method according to any one of claims 1 to 3, for the absolute quantification of the messenger RNAs of one or more cytokines in a biological sample, comprising the following steps: (i) for each of the messenger RNAs of cytokines to be quantified, inverse transcription, then amplification of the product of this reverse transcription, using a specific pair of primers of each messenger RNA, from the biological sample, and from a range of synthetic target RNA comprising the sequence amplified by the specific pair of primers, (ii) the reverse transcription of one or more RNAs of household genes to be quantified, and then the amplification of the product of this reverse transcription, using a pair of d the specific primers of each household gene RNA, from the biological sample, and from a range of synthetic household gene RNAs comprising the sequence amplified by the specific primer pair, s ( iii) real-time detection of the product of the amplification reactions of step (i) and (ii), (iv) deduction, on the one hand, of the amount of RNA or messenger RNAs of cytokines in the sample and on the other hand the amount of RNA or RNAs of household genes in the sample, and (v) Normalization of the amount of RNA or messenger RNAs of cytokines relative to the amount of the RNA or RNA from household genes.   The method according to claim 3 or 4, wherein for each messenger RNA to be quantified, the range of synthetic RNA consists of a series of dilutions of an RNA transcribed from a DNA cassette comprising the promoter. phage T7 RNA polymerase in 5 'and 3' of the amplicon sequence amplified from the messenger RNA, by the specific pair of primers of said messenger RNA.   The method of any one of claims 1 to 5, wherein steps (i) and (ii) are performed by RT-PCR, all RT-PCR reactions being performed under the same temperature conditions.   The method according to any one of claims 1 to 6, wherein the real-time detection of the amplification product is performed by measuring a signal capable of being electronically transcribed, preferably by measuring the incorporation of a fluorescent intercalant, and even more preferably by the incorporation of SYBR Green I. The method of claim 6 or 7, wherein the RT-PCR is carried out in a single step.   The method according to any of claims 1 to 8, wherein step (i) is preceded by a step of introducing a known amount of heterologous RNA or heterologous cells into the biological sample. and a step of extracting the RNA from the biological sample, and wherein said heterologous RNA or the RNA of said heterologous cells is subjected, in step (i), to a reverse transcription followed amplification of the product of this reverse transcription.   The process of any one of claims 6 or 7, wherein the RT-PCR reactions are performed under the following thermocycling conditions: reverse transcription:   incubation of the primers: 8 to 12 minutes at 23 to 27 ° C., preferably 10 minutes at 25 ° C. reverse transcriptase action: 20 to 80 minutes at 45 to 51 ° C, preferably 30 minutes at 48 ° C; inactivation of the reverse transcriptase: 4 to 10 minutes between 90 and 98 ° C., preferably 5 minutes at 95 ° C. 2 to 7 minutes at 45 to 55 ° C., preferably 5 minutes at 50 ° C., then 8 to 12 minutes at 90 ° to 98 ° C., preferably 10 minutes at 95 ° C. then 30 to 45 cycles, preferably 40 cycles, comprising: 12 to 20 seconds between 90 and 98c, preferably 30 seconds at 95 ° C., and 50 to 80 seconds between 55 and 65 ° C., preferably 1 minute. at 60 C.   11. A process according to any one of claims 6 or 7 wherein the RT-PCR reactions are carried out under the following thermocycling conditions: - reverse transcription:   incubation of the primers: 10 minutes at 25 ° C .; reverse transcriptase action: 30 minutes at 48 ° C; inactivation of the reverse transcriptase: 5 minutes at 95 ° C .; 5 minutes at 50 C then 10 minutes at 95 C; then 40 cycles, comprising: 15 seconds at 95 ° C., and 1 minute at 60 ° C.   The method according to any one of claims 1 to 11, wherein at least two of the primer pairs are selected from the following or from the primer pairs having at least 85% identity with the primer pairs following: human IL-1 specific pair: 5'CCTGTCCTGCGCTGTTGAAAGA -3 '(SEQ ID No: 1) and 5'-GGGAACTGGGCAGACTCAAA -3' (SEQ ID No: 2) - specific pair of IL-1 f Human: 5'GCTGGAGGACTTTAAGGGTTACCT -3 '(SEQ ID NO: 3) and 5'CTTGATGTCTGGGTCTTGGTTCT -3' (SEQ ID NO: 4) - specific pair of human IL-12p40: 5'-CATGGTGGATGCCGTTCA -3 '( SEQ ID NO: 5) and 5'ACCTCCACCTGCCGAGAAT -3 '(SEQ ID NO: 6) - specific pair of human IL-13: 5'-ACAGCCCTCAGGGAGCTCAT -3' (SEQ ID NO: 7) and 5'TCAGGTTGATGCTCCATACCAT- 3 '(SEQ ID NO: 8) - specific pair of human IL-15: 5'-CCATCCAGTGCTACTTGTGTTTACTT -3' (SEQ ID No: 9) and 5'CCAGTTGGCTTCTGTTTTAGGAA-3 '(SEQ ID No: 10) - couple specific for human IL-18: 5'-CCTGTCCTGCGCTGTTGAAAGA -3 '(SEQ ID No : II) and 5'GGGAACTGGGCAGACTCAAA -3 '(SEQ ID NO: 12) - specific pair of human IFN-γ: 5'-GTTTTGGGTTCTCTTGGCTGTTA -3' (SEQ ID No: 13) and 5'AAAAGAGTTCCATTATCCGCTACATC -3 '( SEQ ID NO: 14) Human MIF specific pair: 5'-GCCCGGACAGGGTCTACA -3 '(SEQ ID NO: 15) and 5'CTTAGGCGAAGGTGGAGTTGTT-3' (SEQ ID No: 16) - specific human TGF-1 pair : 5'-CAAGGGCTACCATGCCAACT -3 '(SEQ ID NO: 17) and 5'AGGGCCAGGACCTTGCTG -3' (SEQ ID NO: 18) - specific pair of human TNF-α: 5'-CCCCAGGGACCTCTCTCTAATC-3 '(SEQ ID No : 19) and 5'GGTTTGCTACAACATGGGCTACA -3 '(SEQ ID NO: 20) specific pair of human IL-4: 5'-AACAGCCTCACAGAGCAGAAGAC -3' (SEQ ID No: 21) 5'GCCCTGCAGAAGGTTTCCTT-3 '(SEQ ID NO: 20) No: 22) - specific pair of human R2-pglobulin: 5'-AATTGAAAAAGTGGAGCATTCAGA-3 '(SEQ ID No: 23) and 5'-GGCTGTGACAAAGTCACATGGTT-3' (SEQ ID No: 24) - specific pair of the YWHAZ sequence human: 5'-ACTTTTGGTACATTGTGGCTTCAA -3 '(SEQ ID NO: 25) 5'-CCGCCAGGACAAACCAGTAT -3' (SEQ ID No: 26) - specific human ubiquitin C pair: 5'-ATTTGGGTCGCGGTTCTTG -3 '(SEQ ID No: 27) 5'TGCCTTGACATTCTCGATGGT -3' (SEQ ID No: 28) A set of nucleotide primer pairs, wherein at least two primer pairs, preferably three, are selected from the following primer pairs or from the primer pairs having at least 85% identity with the primer pairs. following pairs of primers: - specific pair of human IL-1 f3: 5'CCTGTCCTGCGCTGTTGAAAGA -3 '(SEQ ID No: 1) and 5'-GGGAACTGGGCAGACTCAAA -3' (SEQ ID No: 2) - specific pair of human IL-10: 5'GCTGGAGGACTTTAAGGGTTACCT -3 '(SEQ ID NO: 3) and io 5'CTTGATGTCTGGGTCTTGGTTCT -3' (SEQ ID NO: 4) - specific pair of human IL-12p40: 5'-CATGGTGGATGCCGTTCA -3 '(SEQ ID NO: 5) and 5'ACCTCCACCTGCCGAGAAT -3' (SEQ ID NO: 6) - specific pair of human IL-13: 5'-ACAGCCCTCAGGGAGCTCAT -3 '(SEQ ID No: 7) and 5'TCAGGTTGATGCTCCATACCAT -3 '(SEQ ID NO: 8) - specific pair of human IL-15: 5'-CCATCCAGTGCTACTTGTGTTTACTT -3' (SEQ ID No: 9) and 5'CCAGTTGGCTTCTGTTTTAGGAA-3 '(SEQ ID NO: 10) Specific Torque of Human IL-18: 5'-CCTGTCCTG CGCTGTTGAAAGA-3 '(SEQ ID NO: 11) and 5'GGGAACTGGGCAGACTCAAA-3' (SEQ ID NO: 12) - specific pair of human IFN-γ: 5'-GTTTTGGGTTCTCTTGGCTGTTA-3 '(SEQ ID No: 13) and 5'AAAAGAGTTCCATTATCCGCTACATC-3 '(SEQ ID NO: 14) - specific pair of human MIF: 5'-GCCCGGACAGGGTCTACA -3' (SEQ ID No: 15) and 5'CTTAGGCGAAGGTGGAGTTGTT -3 '(SEQ ID No: 16) specific pair of human TGF-R1: 5'-CAAGGGCTACCATGCCAACT -3 '(SEQ ID No: 17) and 5'AGGGCCAGGACCTTGCTG-3' (SEQ ID No: 18) - specific pair of human TNF-α: 5'-CCCCAGGGACCTCTCTCTAATC -3 '(SEQ ID NO: 19) and 5'GGTTTGCTACAACATGGGCTACA -3' (SEQ ID NO: 20) specific pair of human IL-4: 5'-AACAGCCTCACAGAGCAGAAGAC -3 '(SEQ ID No: 21) 5' GCCCTGCAGAAGGTTTCCTT-3 '(SEQ ID NO: 22) - specific human β-globulin pair: 5'-AATTGAAAAAGTGGAGCATTCAGA-3' (SEQ ID NO: 23) and 5'-GGCTGTGACAAAGTCACATGGTT-3 '(SEQ ID No: 24) , io - specific pair of the human YWHAZ sequence: 5'-ACTTTTGGTACATTGTGGCTTCAA -3 '(SEQ ID No: 25) 5'- CCGCCAGGACAAACCAGTAT -3 '(SEQ ID No: 26) - specific pair of human Ubiquitin C: 5'-ATTTGGGTCGCGGTTCTTG -3' (SEQ ID No: 27) 5'TGCCTTGACATTCTCGATGGT -3 '(SEQ ID No: 28) said at least two pairs of primers being usable under conditions as defined in claim 10 or 11 for quantifying messenger RNAs of one or more cytokines in a biological sample.   14. A set of nucleotide primer pairs, wherein at least two primer pairs, preferably three, are selected from the following primer pairs or from the primer pairs having at least 85% identity with the primer pairs following pairs of primers: - specific pair of human IL-1 R: 5'-CCTGTCCTGCGCTGTTGAAAGA -3 '(SEQ ID No: 1) and 5'GGGAACTGGGCAGACTCAAA -3' (SEQ ID No: 2) - specific pair of human IL-10: 5'-GCTGGAGGACTTTAAGGGTTACCT -3 '(SEQ ID NO: 3) and 5'CTTGATGTCTGGGTCTTGGTTCT -3' (SEQ ID NO: 4) - specific pair of human IL-12p40: 5'- CATGGTGGATGCCGTTCA -3 '(SEQ ID NO: 5) and 5'ACCTCCACCTGCCGAGAAT -3' (SEQ ID NO: 6) - specific pair of human IL-13: 5'-ACAGCCCTCAGGGAGCTCAT -3 '(SEQ ID No: 7) and 5'TCAGGTTGATGCTCCATACCAT -3 '(SEQ ID NO: 8) - specific human IL-15 pairing: 5'-CCATCCAGTGCTACTTGTGTTTACTT-3' (SEQ ID No: 9) and 5'CCAGTTGGCTTCTGTTTTAGGAA-3 '(SEQ ID No : 10) - specific pair of human IL-18: l0 5'- CCTGTCC TGCGCTGTTGAAAGA -3 '(SEQ ID NO: 11) and 5'GGGAACTGGGCAGACTCAAA -3' (SEQ ID NO: 12) - specific pair of human IFN-γ: 5'-GTTTTGGGTTCTCTTGGCTGTTA -3 '(SEQ ID NO: 13) and 5'AAAAGAGTTCCATTATCCGCTACATC -3 '(SEQ ID NO: 14) - specific human MIF pair: 5'-GCCCGGACAGGGTCTACA -3' (SEQ ID No: 15) and 5'CTTAGGCGAAGGTGGAGTTGTT -3 '(SEQ ID No: 16) - specific pair of human TGF-R1: 5'-CAAGGGCTACCATGCCAACT -3 '(SEQ ID No: 17) and 5'AGGGCCAGGACCTTGCTG -3' (SEQ ID No: 18) - specific pair of human TNF-α: 5'-CCCCAGGGACCTCTCTCTATC - 3 '(SEQ ID NO: 19) and 5'GGTTTGCTACAACATGGGCTACA -3' (SEQ ID NO: 20) - specific pair of human IL-4: 5'-AACAGCCTCACAGAGCAGAAGAC -3 '(SEQ ID NO: 21) GCCCTGCAGAAGGTTTCCTT -3 '(SEQ ID NO: 22) - specific pair of human R2-pglobulin: 5'-AATTGAAAAAGTGGAGCATTCAGA-3' (SEQ ID NO: 23) and 5'-GGCTGTGACAAAGTCACATGGTT-3 '(SEQ ID No: 24) ), specific pair of the human YWHAZ sequence: 5'-ACTTTTGGTACATTGTGGCTTCAA -3 '(SEQ ID No: 25) 5'-C CGCCAGGACAAACCAGTAT -3 '(SEQ ID NO: 26) - specific pair of human Ubiquitin C: 5'-ATTTGGGTCGCGGTTCTTG -3' (SEQ ID No: 27) 5'TGCCTTGACATTCTCGATGGT -3 '(SEQ ID No: 28) said at at least two pairs of primers being usable in a method, as defined in claims 4 to 11, for quantifying messenger RNAs of one or more cytokines in a biological sample.   A Thl / Th2 lymphocyte regulatory disorder detection kit comprising a set of primer pairs according to claim 13 or 14, wherein the primer pairs are specific for cytokine messenger RNAs selected from IL-10, IL-13, TGF-13, TNF-α, and IFN-γ.   The detection kit of claim 15, further comprising one or more cytokine-specific messenger RNA primer pairs selected from IL-2, IL-4, IL-5, and TNF. -p.   A kit for detecting inflammatory disorders or innate immunity disorders, comprising a set of primer pairs according to claim 13 or 14, wherein the primer pairs are specific for cytokine messenger RNAs selected from the group consisting of IFN-γ, TNF-α, TGF-f3, IL-1-13, IL-12β40, IL-15 and IL-18 and a pair of mRNA-specific primers IL-12p35.   The detection kit of claim 17, further comprising one or more cytokine-specific mRNA-specific primer pairs selected from IFN-α, IL-6, IL-8, IL. -21, IFN-t3, iNOS and IL-16. n   A kit for the analysis of cytokine expression levels, comprising a set of primer pairs according to claim 13 or 14, wherein a pair of primers is specific for the messenger RNA of IL-10. one or more primer pairs are specific for housekeeping genes selected from the human E32-microglobulin genes, ubiquitin C and the YWMAZ gene, and other primer pairs are specific for messenger RNAs of selected cytokines among IL-19, IL-20, IL-22 and IL-24.   io A kit for the analysis of cytokine expression levels, comprising a set of primer pairs according to claim 13 or 14, wherein a pair of primers is specific for the messenger RNA of IL-12p40. one or more primer pairs are specific for housekeeping genes selected from the human 32 microglobulin genes, ubiquitin C and the YWMAZ gene, and other primer pairs are specific for selected cytokine messenger RNAs. among IL-12p35, IL-23, IL-27 and AK155.   21. Kit according to any one of claims 15 to 20, characterized in that it further comprises, for each specific pair of primers of a messenger RNA, a synthetic RNA comprising the sequence of said messenger RNA as resulting from amplification by the pair of primers, in the form of a solution, a range, or in dry form.   22. Kit according to any one of claims 15 to 20, characterized in that it further comprises, for each specific pair of primers of a messenger RNA, a synthetic DNA comprising the sequence of the complementary DNA such as resulting from amplification by the pair of primers, in the form of a solution, a range, or in dry form.   23. A kit comprising a set of primers according to claim 13 or 14, and a data processing software capable of processing the results obtained at the end of steps (i) to (iii) of a method according to any of claims 4 to 12, and to deduce the amount of messenger RNA from each cytokine tested in the biological sample.   24. Set of primer pairs according to claim 13 or 14, or diagnostic kit according to any one of claims 15 to 23, characterized in that it comprises specific pairs of primers messenger RNAs from at least three, four, five, six, seven, eight, nine or ten cytokines.   25. Set of primer pairs or diagnostic kit according to claim 24, characterized in that it further comprises pairs of specific primers messenger RNAs of at least one, two, three or four household genes.   Kit according to any one of claims 20 to 25, further comprising an RNA that can be used as heterologous RNA, or cells that can be used as heterologous cells during the implementation of the process according to claim 10 and, if appropriate, specific primers of said heterologous RNA or an RNA sequence of said heterologous cells.