EP1651776A2

EP1651776A2 - Method for the reverse transcription and/or amplification of nucleic acids

Info

Publication number: EP1651776A2
Application number: EP04763506A
Authority: EP
Inventors: Tanja Wille; Christian Korfhage; Eric Lader
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2003-07-24
Filing date: 2004-07-26
Publication date: 2006-05-03
Also published as: WO2005010209A2; WO2005010209A3; AU2004259851A1; US20060281092A1; CA2533119A1; JP2006528482A

Abstract

The invention relates to a method for the reverse transcription and/or amplification of a product from a reverse transcription of a pool of nucleic acids of a certain type, whereby said pool of nucleic acids is derived from a complex biological sample or an enzymatic reaction.

Description

Process for the reverse transcription and / or amplification of nucleic acids

The present invention relates to a method for reverse transcription and / or amplification of a product from a reverse transcription of a pool of nucleic acids of a certain type, this pool of nucleic acids originating from a complex biological sample or an enzymatic reaction.

Due to the increasing specificity and sensitivity in the preparation of nucleic acids, this has become increasingly important not only in the field of basic biotechnological research, but also increasingly in medical fields, primarily for diagnostic purposes. Since many molecular biological applications require the separation of certain nucleic acids from one another, the main focus here is on improving and / or simplifying methods for separating and / or isolating nucleic acids. This particularly includes the separation of individual nucleic acid types from complex biological samples and / or from products of enzymatic reactions.

Here, the potential nucleic acid sources are first unlocked using methods known per se. The nucleic acids are then isolated using methods which are also known per se. As a result of such isolation procedures, if further process steps or downstream analyzes such as transcription and / or enzymatic amplification reactions are used, the isolated nucleic acids should not only be free from interfering cell components and / or metabolites. In order to increase the specificity and sensitivity of such applications, an additional purification of individual nucleic acid types is often necessary.

Different single or double-stranded deoxyribonucleic acids (DNA) and / or ribonucleic acids (RNA), such as copy DNA (cDNA), genomic DNA (gDNA), messenger RNA (mRNA), transfer RNA () tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), bacterial DNA, plasmid DNA (pDNA), viral DNA or viral RNA etc., and / or modified or artificial nucleic acids or also nucleic acid analogues, such as peptides nucleic acids (PNA) or Locked Nucleic Acids (LNA) etc., understood. Numerous methods are known for analyzing gene expression patterns, particularly at the level of RNA. In addition to various other methods, reverse transcription reactions with polymerase chain reaction (RT-PCR) and array analysis are among the most frequently used methods. A common feature of these methods is that the mRNA of interest (apart from exceptions, e.g. through direct labeling of RNA) is not measured directly, but is first rewritten into the corresponding cDNA. Current systems, however, already have a fundamental problem at this point when working with biological material, particularly in the field of molecular biology and / or diagnostics.

In order to be able to measure the mRNA (s) of interest in the desired downstream analysis as sensitively as possible, it is also preferred that only this RNA be reversely transcribed. However, since many biological starting materials, such as brain, liver or muscle tissue, whole blood, isolated leukocytes or other biological materials, as well as in products of enzymatic reactions, certain transcripts are present in very large numbers (such as globin mRNA transcripts in RNA preparations from whole blood or rRNA transcripts in all total RNA isolations), these RNA transcripts are also reverse transcribed to a certain degree, for example by unspecific priming and / or mispriming. However, these cDNAs synthesized from the so-called non-mRNA templates and the cDNAs produced from the non-interesting, possibly overexpressed mRNAs cause a significant reduction in the sensitivity of the downstream analyzes of the mRNA (s) of interest.

To avoid unspecific priming and / or mispriming of the non-mRNA templates, commercial oligo-dT primers are frequently used in common methods for priming the reverse transcription, so that only mRNAs that have a poly- Have a tail. Nevertheless, despite the use of oligo-dT primers, unspecific priming and / or mispriming, other types of RNA, such as rRNA, tRNA, snRNA etc., are also reverse-transcribed to a certain degree, so that a reduction is often also found here the sensitivity of the downstream analyzes of the mRNAs cannot be excluded.

This undesired reverse transcription of non-mRNA templates that do not have a poly-A tail is currently often tolerated because of alternative methods of depletion For example, rRNA, tRNA and snRNA transcripts are very complex and cost-intensive, lead to a sequence bias, and often have poor yields.

In addition, many methods require analysis of gene expression patterns at the RNA level, such as. B. Array analyzes, a reverse transcription of the mRNA of interest with a subsequent cDNA double-strand synthesis. This double-strand synthesis is necessary so that the double-stranded cDNA generated in this way can be amplified and / or labeled in a subsequent in vitro transcription (IVT). After this enzymatic reaction has ended, the reaction mixture here also contains the total RNA used and cDNA single strands in which no double strands were synthesized, in addition to the synthesized ds-cDNA. These different single-stranded nucleic acid types are also "carried over" into the subsequent IVT and into the hybridization batch on the array and likewise lead to a reduction in the signals on the array.

In order to increase the sensitivity of such applications, additional purification of DNA with simultaneous depletion of RNA is required. Current methods for depleting RNA from a sample that contains both types of nucleic acid include digestion with RNase. However, the RNase must be added as a separate enzyme for the second strand synthesis in an additional pipetting step, which makes such processes very time-consuming and cost-intensive. In addition, the RNase can only be partially removed completely from the sample.

In order to overcome the disadvantages known from the prior art, the object of the present invention is to provide an efficient method for the selective reverse transcription and / or amplification of the nucleic acid (s) of interest which enables the production of a highly pure nucleic acid from a complex biological sample or Enables an enzymatic reaction that can or can be measured as sensitively as possible in a desired downstream analysis.

This object is achieved according to the invention by a method for reverse transcription and / or amplification of a product of a reverse transcription of a pool of nucleic acids of a type (A) from a biological sample or an enzymatic reaction, characterized by the selective suppression of the reverse transcription of at least one unwanted nucleic acid of type (A) and / or the selective suppression of the amplification of a product of a reverse transcription of at least one undesired nucleic acid of type (A).

The method according to the invention is characterized in particular by the fact that by selectively suppressing the reverse transcription of at least one undesired nucleic acid of a type (A) and / or by selectively suppressing the amplification of a product of a reverse transcription of at least one undesired nucleic acid of a type (A) , which is present in a pool of nucleic acids of type (A) originating from a complex biological sample or an enzymatic reaction, certain nucleic acids of type (A) or amplification products thereof in highly pure form and free of undesired nucleic acids of type (A) or their amplification products be separated.

Biological starting materials in the sense of the invention are complex biological samples, such as tissue samples from neuronal, liver or muscle tissue etc., isolated cells (e.g. leukocytes), whole blood and / or samples contaminated with whole blood (e.g. tissue samples from blood vessels or other highly perfused tissues) as well as other biological materials. Furthermore, the term biological starting materials for the purposes of the invention also includes the products of enzymatic reactions, such as, for example, products of at least one nucleic acid amplification reaction (for example an IVT).

For the purposes of the present invention, the nucleic acid of type (A) is mRNAs, which may be natural mRNAs or those derived from in vitro transcription reactions. Furthermore, the term “undesired nucleic acid of type (A)” in the context of the invention is understood to mean at least one mRNA which in each case accounts for a share of 20% or more in the total mRNA. As already mentioned above, certain undesired mRNAs can be found in samples certain starting materials may be present in very high numbers of copies, such as globin mRNAs in RNA isolated from whole blood, cytochrome mRNAs in RNA isolated from muscle cells or myelin mRNAs in RNA isolated from neuronal tissue. The proportion of these mRNA (s) can do more here account for more than 40% or even more than 60% of the total mRNA.

Surprisingly, it was found that the method according to the invention efficiently suppressed the reverse transcription of at least one undesired one Nucleic acid of a type (A), and / or the amplification of a product of a reverse transcription of at least one undesired nucleic acid of a type (A), in particular globin mRNA. Regardless of whether the whole blood sample was taken promptly or was taken up and stored in a stabilizing reagent.

Advantageously, the blood samples used in the method according to the invention are converted into a stabilizing reagent immediately upon acceptance to maintain the RNA status. Known compounds such as tetra-alkyl ammonium salts in the presence of an organic acid (WO 02/00599 / QIAGEN GmbH, Hilden, DE) or guanidine compounds in a mixture with a buffer substance, a reducing agent and / or a detergent (e.g. WO 01/060517 / Antigen Produktions GmbH, Stuttgart, DE) can be used. Such a procedure enables blood collection tubes in which the stabilizing reagent is already contained (PaxGene / PreAnalytix, Hombrechticon, CH).

To carry out the method according to the invention, the individual method steps can also be designed differently. However, the method according to the invention is based on method step a), carrying out a reverse transcription reaction of an RNA from a biological sample or an enzymatic reaction in the presence of at least one oligo-dT primer. Optionally, subsequent to a), process steps b), carrying out a cDNA second-strand synthesis, and c), purifying the ds-cDNA formed in b) with simultaneous depletion of all single-stranded nucleic acids from the reaction product of b). Furthermore, according to a) and / or b) and / or c) an amplification of the cDNA can be carried out.

According to a first embodiment of the method according to the invention, the first method step (a) is carried out according to methods known per se in the prior art with common reagents, such as, for example, a commercial reverse transcriptase (e.g. Superscript II RT / Invitrogen) and in the presence of at least one commercially available oligo-dT Primers (T7-oligo-dT ₂ Primer / Operon, Cologne, DE).

As already mentioned at the beginning, common methods for reducing the reverse transcription of the nucleic acids different from type (A) for priming the reverse transcription frequently use commercially available oligo-dT primers or derivatives and / or fusions from oligo-dT primers, such as, for example, primers with sequences for a T7 RNA polymerase promoter at the 5'-end and oligo-dT sequences at the 3'-end, so that only mRNAs which have a poly-A sequence at the 3'-end are preferably reverse transcribed. For the purposes of the invention, the nucleic acids different from type (A) are essentially different RNA types than mRNAs (e.g. rRNA, tRNA, snRNA, gDNA and plastid DNA), the so-called non-mRNA templates ,

Subsequent to process step a), a cDNA second-strand synthesis can then optionally be carried out using a method known per se, including the common reagents. For example, an RNase H is added as a separate enzyme before the start of the second strand synthesis, the mRNA hybridized to the cDNA after the first strand synthesis being degraded by the activity of the enzyme (while the non-hybrid RNA is not a substrate for the RNase H). The reaction is carried out in such a way that the RNase H digestion is incomplete, so that shorter RNA fragments remain. These RNA fragments serve as primers for the subsequent second strand synthesis.

To avoid additional pipetting steps and to save material, etc., in a preferred embodiment of the method according to the invention, a specific reverse transcriptase (e.g. LabelStar RT / QIAGEN GmbH, Hilden, DE) is advantageously used which has an intrinsic RNase H activity has, so that the cDNA second strand synthesis can be carried out much faster, easier and cheaper (see Example 1).

After this enzymatic reaction has ended, in addition to the synthesized ds-cDNA, the reaction mixture usually also contains the total RNA used and cDNA single strands (e.g. ss cDNA, viral cDNA etc.) on which no double strands were synthesized (inter alia because the synthesis of the double strand does not happen with 100% efficiency). These different nucleic acid types are also "carried over" into a subsequent amplification reaction and / or thus into the hybridization batch on the array without an effective purification step. During the hybridization, the various unlabeled nucleic acids in solution compete for the binding with the labeled cRNA transcripts to the probes on the array Furthermore, the probes on the array compete with the unlabeled nucleic acid transcripts in solution for binding to the labeled ones cRNAs instead. Since the balance of these competitive reactions is not entirely on the side of the hybridization of the labeled cRNAs to the probes on the array, the presence of the unlabeled nucleic acids leads to a reduction in the signals on the array.

The unwanted hybridization of one or more overrepresented labeled or unlabeled nucleic acid transcripts to the probes on the array can be further reduced by adding unlabeled oligonucleotides that have the reverse complementary sequence to the undesired nucleic acid transcripts. These reversely complementary oligonucleotides can be, for example, oligonucleotides transcribed in vitro or synthetically produced. The consequent reduction in the unspecific hybridization of overrepresented transcripts increases the sensitivity of the array analysis.

In order to avoid the "carryover" of the different nucleic acid types, a conventional purification of the reaction mixture of the enzymatic reaction can take place after step b). The actual purification step takes place, for example, by using "silica spin column technologies" known in the art. (eg with the commercially available GeneChip Sample Cleanup Module / Affymetrix, Santa Clara, US). After the addition of a binding buffer containing chaotropic salts, the reaction mixture is separated using a commercially available spin column (eg MinElute Cleanup Kit / QIAGEN GmbH, Hilden, DE). However, since this often leads to contamination of the eluate by "carried over" RNA from the total RNA, RNase digestion is carried out in conventional purification processes to eliminate the total RNA used from the sample. However, the RNase digestion is very costly and time-consuming due to the additional material and the additional work steps. In addition, the RNase cannot subsequently always be completely removed from the sample, which, for example in the case of a subsequent amplification in which the sample is brought into contact with RNA, disadvantageously leads to the degradation of this RNA.

Surprisingly, it was found that the RNase digestion becomes superfluous due to an additional washing step following the binding of the different nucleic acids to the column material. Process step c) according to the invention can not only advantageously replace an upstream isolation of mRNA, but at the same time it enables the depletion of all single-stranded nucleic acids (ss DNAs and RNAs) from the reaction product from step b), with purification of the ds-cDNA. Furthermore, the use of the washing step according to the invention enables the production of a ds-cDNA with a high degree of purity, which leads to an enormous increase in sensitivity in a subsequent GeneChip analysis (see Example 10).

In addition to the depletion of single-stranded RNA and cDNA, at least one single-stranded nucleic acid transcript can also be separated from other single-stranded transcripts by using the washing step according to the invention. Here, the oligonucleotides which are complementary to the single-stranded target sequence and which form a double-stranded nucleic acid hybrid with the target sequence are used. In the case of subsequent purification using the washing step according to the invention, all non-hybridized, and thus still single-stranded, transcripts are separated from the nucleic acid mixture.

To purify the ds-cDNA according to the inventive method in step c), the nucleic acids originating from step b) are first bound in their entirety to a silica matrix and then the silica matrix is washed with a guanidine-containing washing buffer to deplete the single-stranded nucleic acids. If the reverse transcription of the total RNA was primed with oligo-dT primers, cDNA molecules were primarily synthesized which are complementary to the mRNA molecules of the starting RNA (ie no cDNA synthesis based on rRNA, tRNA, snRNA molecules) , After the reaction solution has been added to the silica spin columns, or added to the silica particles, the method described above allows the depletion of all single-stranded nucleic acids in one washing step with a washing buffer according to the invention.

The washing step according to the invention can advantageously be used in any process in which purification of double-stranded nucleic acids with simultaneous depletion of single-stranded nucleic acids is desired. The washing step according to the invention can thus also take place after the optional process step d) shown below (carrying out an amplification of the cDNA).

The silica matrix used for the purification can comprise one or more silica membrane (s) or particles with a silica surface, in particular magnetic silica particles, and can be contained in a spin column or other common devices for nucleic acid purification. The guanidine-containing washing buffer used for the washing step according to the invention preferably contains guanidine isothiocyanate and / or guanidine thiocyanate, preferably in a concentration of 1 M to 7 M, particularly preferably from 2.5 M to 6 M and very particularly preferably from 3 M to 5.7 M. As an alternative to guanidine isothiocyanate and / or guanidine thiocyanate, guanidine hydrochloride can also be used according to the invention, with a concentration of 4 M to 9 M, preferably 5 to 8 M.

As further constituents, the washing buffer used in the washing step according to the invention can contain one or more buffer substance (s) in a total concentration of 0 mM to

40 mM and / or one or more additive (s) in a total concentration of 0 mM to

100 mM and / or one or more detergent (s) in a total concentration of

0% (v / v) to 20% (v / v) included.

The pH of the washing buffer is preferably in the range from pH 5 to 9, particularly preferably in the range from pH 6 to 8, with common buffer substances (such as Tris, Tris-HCl, MOPS, MES, CHES, for example) for setting the corresponding pH , HEPES, PIPES and / or sodium citrate), preferably with a total concentration of the buffer substances 20 mM to 40 mM, can be used.

Furthermore, depending on the individual reaction conditions, other suitable additives, such as, for example, chelating agents (for example EDTA, EGTA or other suitable compounds) and / or detergents (for example Tween 20, Triton X 100, Sarcosyl, NP40 etc.) Wash buffer composition can be added.

The following list shows preferred compositions of the washing buffer used in the washing step according to the invention:

- Wash buffer 1: 3.5 M guanidine isothiocyanate ^* 25 mM sodium citrate, pH 7.0

- Wash buffer 2: 5.67M guanidine isothiocyanate * 40 mM sodium citrate pH 7.5 - Wash buffer 3: 5.0M guanidine isothiocyanate ^* 35 mM sodium citrate pH 7.5

- Wash buffer 4: 4.5 M guanidine isothiocyanate ^* 32 mM sodium citrate pH 7.5

- Wash buffer 5: 4.0 M guanidine isothiocyanate ^* 28 mM sodium citrate pH 7.5

- Wash buffer 6: 3.5 M guanidine isothiocyanate ^* 25 mM sodium citrate pH 7.5

Wash buffer 7: 4.5 M guanidine isothiocyanates ^* 0.1 M EDTA, pH 8.0

Wash buffer 8: 7.0 M guanidine hydrochloride, pH 5.0

Wash buffer 9: 5.6 M guanidine hydrochloride 20% Tween-20

* Guanidine thiocyanates can be used in combination with or instead of guanidine isothiocyanates.

The use of the washing step according to the invention described above can thus be used to deplete rRNA from double-stranded eukaryotic cDNA synthesis products. Another application is the separation of single-stranded viral nucleic acids from eukaryotic or prokaryotic, double-stranded genomic DNA (see Example 4).

As already mentioned, the washing step according to the invention for the depletion of single-stranded nucleic acids from double-stranded nucleic acids is advantageous for various downstream analyzes. In addition to array analyzes, the sensitivities could also be increased, for example in amplification reactions or other applications (such as, for example, ribonuclease protection assays, Northern or Southern blot analyzes, primer extension analyzes, etc.). Surprisingly, it was found that, on the one hand, the implementation of individual process steps of the process according to the invention already improves the purity of the nucleic acid of interest obtained from the different samples, but on the other hand, in particular, the combination of individual process steps in different ways produces synergetic effects which lead to the production of at least one highly pure nucleic acid of type (A) contributes.

In addition to increasing the specificity by specifically priming cDNA syntheses with a corresponding reverse transcriptase, an unwanted high number of existing mRNA transcripts, such as globin mRNA transcripts from a whole blood sample, for subsequent downstream analyzes can also be caused by the presence of a molecule Species to suppress an RT and / or amplification reaction of the unwanted mRNA transcripts are eliminated.

Thus, according to a further advantageous embodiment of the present invention, the process steps a) and / or d) in the presence of at least one molecular species for the selective suppression of the reverse transcription of at least one undesired mRNA and / or for the selective suppression of the amplification of those from the undesired one or more mRNA (s) produced single or double-stranded cDNA (s), are carried out.

In process step a), the molecular species bind to the unwanted nucleic acids of type (A) or cleave them, thereby preventing the reverse transcription of the unwanted mRNAs.

Amplification in the sense of the invention includes various reaction types, such as in vitro transcription, a polymerase chain reaction (PCR), a ligase chain reaction (LCR), a nucleic acid sequence-based amplification (NASBA) or a self-sustained sequence replication (3SR) etc. understood.

Depending on the type of biological sample or the enzymatic reaction product, it may be advantageous to use the molecular species both in process step a) and subsequently in process step d). The molecular species used can be the same or different in all process steps. According to a further preferred embodiment of the method according to the invention, method step a) is therefore carried out in the presence of at least one molecule species for the selective suppression of the reverse transcription of at least one undesired mRNA, the reverse transcription of the overrepresented transcripts being interrupted by binding of the molecule species to these mRNAs becomes. These transcripts are therefore no longer available for cDNA labeling, double-strand synthesis and / or the subsequent amplification.

Molecular species within the meaning of the invention can be complementary to the mRNA or to one of the cDNA strands DNA or RNA oligonucleotides (antisense oligonucleotides) or their derivatives, e.g. B. oligonucleotides containing modified or artificial nucleotides, quenchers, fluorophores or other modifications, in a length of 10 to 60 nucleotides, preferably 12 to 30 nucleotides.

In addition, the molecule species can be a nucleic acid analogue which is complementary to the mRNA or to one of the cDNA strands, with modified nucleic acids, such as PNAs (peptide nucleic acids), LNA (locked nucleic acids), and / or GripNAs also being used as the nucleic acid analogue can be used. The molecule species used for sequence-specific blocking binds preferably in the 3 'region of the nucleic acid to be blocked (the mRNA or one of the cDNA strands).

Preferred molecular species are PNAs that have a length of 12 to 20 nucleotide analogs, preferably 13 to 16 nucleotide analogs (PE Biosystems, Weiterstadt, DE) and / or GripNAs that have a length of 12 to 30 nucleotide analogs, preferably 14 to 20 Have nucleotide analogs (ActiveMotif) and / or LNAs which have at least one nucleotide which is a 'locked nucleotide' and which have a length of 14 to 30 nucleotides, preferably 15 to 22 nucleotides (Operon, Cologne, DE) , used.

As an alternative to using a single molecule for sequence-specific blocking of a specific target sequence, it is also possible to use several molecules complementary to different regions within one or more specific target sequence (s). Furthermore, it can also prove to be advantageous to use a single molecule for sequence-specific blocking which acts against several different target RNAs or target cDNAs if the molecule is complementary to a homologous region of different target RNAs or target cDNAs.

If the molecule species which is used for sequence-specific blocking is used, for example, to prevent nucleic acid polymerization (for example an RT), this molecule species must have a modification at its 3 'end (for example by acetylation, phosphorylation, carboxylation or other suitable modifications) which prevent the molecular species from not serving as primers themselves and consequently initialize an elongation beginning at the 3 'end of the molecular species. In an alternative embodiment of the invention, the labeling of RNA is prevented by hybridizing the RNA with tightly binding molecules.

As an alternative to blocking the target sequence, as already mentioned at the beginning, there is also the possibility of cleaving specific unwanted or undesired mRNAs in a sequence-specific manner by means of certain molecular species. Molecular species such as DNAzymes, ribozymes, in particular hammerhead ribozymes and / or hairpin ribozymes, can be used for this purpose. These molecules are preferably directed against the 3 'region of the unwanted RNA and are used before the reverse transcription is carried out. Ribozymes consisting of RNA or RNA derivatives or fusion products of such ribozymes can be used for this embodiment of the invention. The complementary sequence of the ribozymes preferably has a length of 12 to 30 nucleotides, particularly preferably a length of 15 to 25 nucleotides.

According to the invention, one or more DNA oligonucleotide (s), PNA (s) and / / are advantageously used as molecular species for selective suppression or for blocking the reverse transcription or amplification of the undesired mRNA, in particular the globin sequences. or LNA (s) which have the sequences listed below are used.

If the molecular species is a DNA oligonucleotide and the globin mRNA is an alpha 1-globin mRNA and / or an alpha 2-globin mRNA, the DNA oligonucleotide has the function of blocking the reverse transcription of globin mRNA invention according to a sequence from the following group which is complementary to human alpha 1 globin mRNA and / or alpha 2 globin mRNA. alpha_473: 5 ^' CTC GAG CTT AAC GGT - phosphate group - 3' alpha_465: 5TAA CGG TAT TTG GAG - phosphate group - 3 'alpha_ 465_long: 5 ^' TAA CGG TAT TTG GAG GTC AGC ACG GTG CTG - phosphate group - 3 '

If the molecule species is a DNA oligonucleotide and the globin mRNA is a beta globin mRNA, the DNA oligonucleotide has a sequence from the following group according to the invention for blocking the reverse transcription of globin mRNA: is complementary to human beta globin mRNA. beta_554: 5 ^' GTA GTT GGA CTT AGG - phosphate group - 3 ^' beta_594: 5 ^' ATC CAG ATG CTG AAG - phosphate group - 3 ^' beta_554_long: 5 ^' GTA GTT GGA CTT AGG GAA CAA AGG AAC CTT - phosphate group - 3 ^'

If the molecular species is a PNA and the globin mRNA is an alpha 1 -globin mRNA and / or an alpha 2-globin mRNA, the PNA has one according to the invention for blocking the reverse transcription of globin mRNA Sequence from the following group, which is complementary to human alpha 1 globin mRNA and / or alpha 2 globin mRNA. alpha_473: N- CTC CAG CTT AAC GGT -C ^* alpha_465: N- TAA CGG TAT TTG GAG -C ^* alpha_363: N- GTC ACC AGC AGG CA -C ^* alpha_393: N- GTG AAC TCG GCG -C ^* alpha_473 ^** : N- TGG CAA TTC GAC CTC -C ^* alpha_465 ^** : N- GAG GTT TAT GGC AAT -C * alpha_363 **: N- ACG GAC GAC CAC TG -C ^* alpha_393 ^** : N- GCG GCT CAA GTG - C ^* If the molecular species is a PNA and the globin mRNA is a beta globin mRNA, the PNA has a sequence from the following group according to the invention for blocking the reverse transcription of globin mRNA, which is complementary to human beta Globin mRNA. beta-554: N- GTA GTT GGA CTT AGG -C ^* beta-594: N- ATC CAG ATG CTC AAG -C ^* beta-539: N- CCC CAG TTT AGT AGT -C * beta-541: N- CAG TTT AGT AGT TGG -C ^* beta-579: N- GCC CTT CAT AAT ATC -C ^* beta-554 ^** : N- GGA TTC AGG TTG ATG -C * beta-594 **: N- GAA CTC GAT GAC CTA - C ^* beta-539 **: N- TGA TGA TTT GAC CCC -C ^* beta-541 ^** : N- GGT TGA TGA TTT GAC -C ^* beta-579 ^** : N- CTA TAA TAG TTC CCG -C ^*

Where N indicates the amino terminus of the oligomers and C * the carboxy terminus of the oligomers, and the sequences provided with (**) are reverse-oriented to the above sequences.

If the molecular species is an LNA which has at least one nucleotide which is a locked nucleotide ¹ , and the globin mRNA is an alpha 1 globin mRNA and / or an alpha 2 globin mRNA, according to the invention, the LNA for blocking the reverse transcription of globin mRNA has a sequence from the following group which is complementary to human alpha 1 -globin mRNA and / or alpha 2-globin mRNA. alpha_473: 5 ^' CTC CAG CTT AAC GGT - octane diol - 3 ^' alpha_465: 5 TAA CGG TAT TTG GAG - octane diol - 3 ^' alpha_363: 5 ^' GTC ACC AGC AGG CA - octane diol - 3 ^' alpha_393: 5 ^' GTG AAC TCG GCG - Octane diol - 3 ^'

If the molecular species is an LNA which has at least one nucleotide which is a 'locked nucleotide', and the globin mRNA is a beta globin mRNA, according to the invention the LNA for blocking the reverse transcription of globin mRNA has a sequence from the following group which is complementary to human beta globin mRNA. beta-554: 5 ^' GTA GTT GGA CTT AGG - octane diol - 3 ^' beta-594: 5 ^' ATC CAG ATG CTC AAG - octane diol - 3 ^' beta-539: 5 ^' CCC CAG TTT AGT AGT - octane diol - 3 ^' beta 541: 5 'CAG TTT AGT AGT TGG - octane diol - 3 ^' beta-579: 5 ^' GCC CTT CAT AAT ATC - octane diol - 3 ^'

In the LNA sequences mentioned, individual or all positions in the oligonucleotides can be substituted with the so-called “locked nucleotides”. These “locked nucleotides” are primarily enzymatically non-degradable nucleotides, which, however, do not serve as a starting molecule for a polymerase can, since they have no free 3'-OH end.

If RNA preparations which have a high proportion of overrepresented transcripts (e.g. globin mRNA transcripts) are reverse transcribed in the presence of the molecular species shown above and / or the products of the reverse transcription are amplified (preferably by in vitro transcription, optionally) with a subsequent DNase digestion and a cRNA purification), and / or if at least one washing step according to the invention is carried out, there are advantageously no RT products or amplification products resulting therefrom, which significantly increases the sensitivity of the gene expression analysis of low or low expressed transcripts can be.

In particular, the use of the cRNA and / or cDNA resulting from the method according to the invention in an array-based gene expression analysis is extremely advantageous since no RT products result from highly expressed transcripts and / or amplification products from RT products of highly expressed transcripts on the arrays are hybridized and thus a reduction in the signal intensities and the associated loss of sensitivity in the array analysis is avoided. The present invention will now be explained in more detail below with reference to the accompanying drawings and the exemplary embodiments below.

It shows:

1 shows the influence of different final concentrations of alpha_465 and beta_554 PNAs on the generation of cRNAs as a graphic representation of the cRNA analysis on the Agilent 2100 bioanalyzer as on a gel, with:

Band L: RNA size standard, band 1: generated cRNA at a final PNA concentration of 10 μM, band 2: generated cRNA at a final PNA concentration of 1.0 μM, band 3: generated cRNA at a final PNA concentration of 0, 1 μM, band 4: generated cRNA at a final PNA concentration of 0.01 μM, band 5: generated cRNA at a final PNA concentration of 0.001 μM band 11: generated cRNA without addition of PNAs (comparative sample).

2 shows the influence of different final concentrations of alpha_465 and beta_554 PNAs on the generation of cRNAs. Presented as an electropherographic image of the cRNA analysis on the Agilent 2100 bioanalyzer, with the curves:

Turquoise (1): generated cRNA without adding PNAs (comparative sample) Yellow (2): generated cRNA at a final PNA concentration of 0.001 μM. Pink (3): generated cRNA at a final PNA concentration of 0.01 μM each. Braun (4): generated cRNA at a final PNA concentration of 0.1 μM. Dark blue (5): generated cRNA at a final PNA concentration of 1.0 μM each. Green (6): generated cRNA at a final PNA concentration of 10 μM.

3 shows the correlation of the signal intensities of the sample in which only Jurkat RNA was used with that of the sample in which Jurkat RNA was analyzed with added globin in vitro transcripts. Fig. 4 shows the amount of RNA in a sample before and after purification with different washing conditions.

Fig. 5 amount of single-stranded cDNA of a sample before and after purification with different washing conditions.

6 shows the presence of RNA and gDNA before and after purification (with different washing conditions) on a formaldehyde agarose gel, where:

Band 1: the genomic DNA (before cleanup); Band 2: the RNA (before cleanup); Band 3: the genomic DNA mixed with the RNA (before the cleanup); Lanes 4 and 5: the genomic DNA mixed with the RNA (after the cleanup); (Purification was carried out under standard conditions using an ethanol-containing washing buffer) Lanes 6 and 7: the genomic DNA mixed with the RNA (after the cleanup); (The purification was carried out under standard conditions with an additional washing step with a washing buffer 1 containing chaotrophic salts).

Ausführunαsbeispiele

Example 1 :

Using the PAXgene Blood RNA Isolation Kit (PreAnalytix, Hombrechticon, CH), RNA was isolated from whole human blood. Subsequently, a gene expression analysis was carried out using the Affymetrix U133A GeneChips. The target preparation was carried out according to the “Expression Analysis Technical Manual” for Affymetrix GeneChip analyzes (Affymetrix, Santa Clara, US). However, two different reverse transcriptases were used in two approaches. Approach 1: was carried out in accordance with the Affymetrix "Expression Analysis Technical Manual" with Superscript II RT (Invitrogen) as reverse transcriptase; and

Approach 2: was also carried out according to the Affymetrix "Expression Analysis Technical Manual", but with 1 / I from LabelStar RT (QIAGEN GmbH, Hilden, DE) as reverse transcriptase. In addition, the reaction buffer belonging to LabelStar RT was also used for cDNA second strand synthesis.

6 μg of the isolated RNA per approach were reversely transcribed from an oligo-dT-T7 primer (Operon, Cologne, DE). The cDNA second strand synthesis and all further steps of sample preparation for the GeneChip analysis were also carried out according to the instructions of the Affymetrix "Expression Analysis Technical Manual", whereby the two different approaches were treated identically. The samples were then hybridized to Affymetrix U 133 A GeneChips To compare the results of the two approaches, both arrays were scaled to TGT = 1000 with the same signal intensities.

Then both approaches were processed with the cDNA Cleanup of the GeneChip Sample Cleanup Module AHx in accordance with the manufacturer's Technical Manual (for further information see Example 12).

The results listed in Table 1 below show that by using the LabelStar reverse transcriptase (specific priming of the cDNA synthesis), the proportion of genes rated as "present" on the gene chip from 34.7% to 39% (by 12%) rise.

Table 1: Results of a GeneChip analysis on U133A GeneChips using different reverse transcriptases. By using the LabelStar reverse transcriptase for the first strand synthesis of the cDNA, the signal intensities for the ribosomal RNA transcripts (18S rRNA and 28S rRNA) could be greatly reduced. Priming with the LabelStar RT as a reverse transcriptase is therefore much more specific for mRNA.

This depletion of the rRNAs also results in a lower scaling factor and a higher rate of "present calls" on the array. (The scaling factor for the sample with the LabelStar RT reverse transcriptase is approximately 50% lower than for the sample with the the SuperScript was reverse transcribed.)

Example 2:

RNA was isolated from whole human blood. The following cDNA synthesis was carried out as in Example 1 with two different reverse transcriptases (SuperScript RT and LabelStar RT) starting from oligo-dT-T7 primers. The cDNA second-strand synthesis was then carried out under identical conditions for the different batches. After purification of the reactions, an IVT was carried out with subsequent purification of the cRNA including a DNase digestion. The DNase digestion ensures that only the generated RNA and not the contaminating cDNA is measured in the subsequent TaqMan RT-PCR analysis (QIAGEN GmbH, Hilden, DE) of the cRNA.

Two different TaqMan RT-PCR analyzes were then carried out:

- quantification of the 18S rRNA - quantification of the p16 mRNA (exemplary for all mRNA transcripts)

It was shown that when using the LabelStar RT, the quantified amount of 18S rRNA was about 8 times lower than when using the Superscript RT. The amount of p16 mRNA quantified, however, is comparable for both reverse transcriptases.

This means that the use of the LabelStar RT specifically depletes the rRNA, while the mRNA transcripts are reverse transcribed with identical efficiency. Example 3:

The RNA of a blood donor was isolated as in Example 1 using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH). In preparation for the subsequent Affymetrix GeneChip analysis, the Affymetrix target preparation was carried out according to the Affymetrix "Expression Analysis Technical Manual" (standard protocol). This approach was compared to a second approach in which the conditions during annealing of the cDNA primer were varied:

Conditions for annealing the cDNA primer annealing:

- Standard protocol: Incubation for 10 min at 70 ° C Rapid cooling on ice Subsequently cDNA synthesis at 42 ° C

- Comparative approach: Incubation for 10 min at 70 ° C Incubation for 5 min at 45 ° C Incubation for 2 min at 42 ° C Following cDNA synthesis at 42 ° C

The subsequent GeneChip analysis on Affymetrix U133A gene chips showed the following results listed in Table 2: (scaling of the signal intensities to TGT = 1000):

Table 2: Results of a GeneChip analysis on U133A GeneChips

The changed conditions during the attachment of the cDNA primer lead to reduced signal intensities for the ribosomal RNAs. Example 4:

The RNA of a blood donor was isolated as in Example 1 using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH). To block the reverse transcription of the globin transcripts (mRNAs), the following PNA sequences (PE Biosystems) were added, which are complementary to the 3 ^' regions of the globin transcripts.

PNA sequence, complementary to human alpha 1 globin mRNA and alpha 2 globin mRNA: alpha_465: N- TAA CGG TAT TTG GAG -C ^*

PNA sequence complementary to human beta globin mRNA: beta_554: N- GTA GTT GGA CTT AGG -C ^*

5 μg RNA were used for reverse transcription per approach. The cDNA synthesis was carried out according to the manufacturer's instructions in the Technical Manual (Affymetrix "Expression Analysis Technical Manual"), the PNA sequences listed above which were complementary to the alpha and beta globin transcripts being additionally added. Before starting the cDNA synthesis, both PNAs (alpha_465 and beta_554) and the primers were incubated in a conventional cDNA synthesis reaction buffer (buffer from Superscript RT / Invitrogen) for 10 min at 70 ° C. and subsequently for 5 min at 42 ° C. Before the reverse transcriptase was added, the PNAs were added in a final concentration of 0.001 μM, 0.01 μM, 0.1 μM, 1, 0 μM and 10 μM. Subsequently, all other components required for the RT (such as additional reaction buffer, nucleotides, dithiothreitol (DTT) and the reverse transcriptase) were added and the samples were incubated at 42 ° C. for 1 h. Both the cDNA double strand synthesis, as well as the in vitro transcription and the cleanup of the cRNA were carried out according to the manufacturer's instructions in the Affymetrix "Expression Analysis Technical Manual". The comparison and control samples without PNAs were treated identically.

After the cRNA was cleaned up, the samples were analyzed on an Agilent 2100 bioanalyzer (Agilent, Böblingen, DE). The corresponding results can be seen in FIGS. 1 and 2. They show the influence of alpha_465 and beta_554 PNAs on the generation of cRNAs, whereby it also becomes clear that the addition of alpha and beta Globin transcripts of complementary PNA oligomers lead to a reduction in the cRNA fragments, which result in a clear band when analyzed on the Agilent 2100 bioanalyzer. These cRNA fragments were generated from the globin transcripts (mRNA) of the starting materials (whole blood). The extent of the reduction depends on the concentration of the PNAs.

Example 5:

The RNA of a blood donor was isolated from human whole blood (without erythrocyte lysis) as in Example 1 using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH). For each approach, 1.7 μg RNA was used in a reverse transcription. The cDNA synthesis was carried out with the reverse transcriptase Omniscript (QIAGEN GmbH, Hilden, DE) according to the manufacturer's instructions (except that the RT was carried out at 42 ° C. instead of 37 ° C.). The cDNA synthesis was primed with a T7-oligo-dT ₂ primer (Operon, Cologne, DE). Before the reverse transcriptase was added, PNAs (sequences see below) were added at a final concentration of 0.5 μM, 1.0 μM and 1.5 / M and the mixture was incubated first at 70 ° C. for 10 min and then at 37 ° C. for 5 min , The reverse transcriptase was then added and the samples were incubated at 42 ° C. for 1 h. The comparison and control samples without PNAs were treated identically.

Following cDNA synthesis, TaqMan PCR reactions were carried out in which the amount of alpha and beta globin cDNA was quantified using a standard series.

Identical primers were used for the amplification of alpha 1 -globin cDNA transcripts and alpha 2-globin cDNA transcripts. The following PNA sequences were used to block the reverse transcription of the alpha and beta globin transcripts:

Sequences that are complementary to human alpha 1 globin mRNA and alpha 2 globin mRNA:

alpha_473: N- CTC CAG CTT AAC GGT -C ^* alpha_465: N- TAA CGG TAT TTG GAG -C ^* Sequences that are complementary to human beta globin mRNA:

beta_554: N- GTA GTT GGA CTT AGG -C ^* beta 594: N- ATC CAG ATG CTC AAG -C ^*

Table 3: Influence of PNAs complementary to alpha and beta globin on a two-step RT-PCR reaction.

The results listed in Table 3 show that the use of the PNAs alpha_473 and / or alpha_465 leads to a reduction in the cDNA amount of the alpha globin transcripts by more than 95%. The transcript level of beta globin remains unaffected when the PNA alpha_473 is used if the final PNA concentration is not higher than 0.5 μM.

The use of the PNAs beta_554 and beta_594 leads to a reduction in the cDNA amount of beta globin by approximately 99% and 80%, respectively. If these PNAs are used in a final concentration of 0.5 μM, the transcript level for alpha globin remains unaffected.

Example 6:

RNA was isolated from two different blood donors using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH). For the subsequent gene expression analysis with Affymetrix U133A gene chips, the target samples for the RNA samples of both donors were prepared using the following protocol variants:

1. Standard protocol (according to Affymetrix Expression Analysis Technical Manual)

2. Target preparation using PNAs to block the reverse transcription of the globins: In comparison to the standard protocol, the following protocol changes were carried out in the batches using the PNAs: The PNAs were primed together with the T7-oligo (dT) ₂₄ before the cDNA synthesis (Operon, Cologne, DE) pipetted to RNA. Several incubation steps were carried out to attach the primer and the PNAs to the RNAs (10 min 70 ° C; 5 min 45 ° C; 2 min 42 ° C). All further steps were carried out as in the standard protocol. Approaches were compared using different PNA combinations and PNA concentrations.

The following PNA combinations and final PNA concentrations (during the annealing reaction) were used for each of the total RNA isolates:

Table 4: PNA combinations and final concentrations

After target preparation, the gene expression analysis was carried out using Affymetrix U133A arrays. For the evaluation, all array data were scaled to signal intensities of TGT = 500. Table 5: Evaluation of all array data after gene expression analysis on Affymetrix U133A arrays

Using the PNAs, the globin signal intensities on the arrays could be reduced by 40-60%. Furthermore, the proportion of genes that were rated "present" on the array was increased from approximately 32% to approximately 43%.

Example 7:

RNA was isolated from whole human blood using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH). During the target preparation for the Affymetrix GeneChip analysis, the PNA was used to block the cDNA synthesis of alpha globin mRNA Oligonucleotide alpha_465 used. During the attachment of the PNAs to the globin mRNA transcripts, two different conditions were compared:

- the starting RNA, the T7-oligo (dT) ₂₄ primer and the PNA oligonucleotide were in water - the starting RNA, the T7-oligo (dT) ₂₄ primer and the PNA oligonucleotide were in 3.5 mM (NH ₄ ) 2SO ₄ before

The subsequent GeneChip analysis using Affymetrix U133A arrays showed that the addition of the PNA oligonucleotide in the presence of ammonium sulfate leads to an increase in the "present call" rate from 40.7% to 42.8%.

Example 8:

RNA was isolated from Jurkat cells (cell line; acute lymphoblatic leukemia). In vitro transcripts were spiked into this RNA, which correspond to the alpha-1 globin, alpha-2 globin and beta globin mRNA sequences. These in vitro transcripts had a poly-A sequence at the 3 ^' end, so that, like naturally occurring mRNA transcripts, they could be rewritten into cDNA by priming with a T7-oligo (dT) ₂ primer. Three different approaches were compared:

1. Jurkat RNA 2. Jurkat RNA with spiked globin in vitro transcripts 3. Jurkat RNA with spiked globin in vitro transcripts using peptide nucleic acids (PNAs) to block globin cDNA synthesis

PNAs used in the third reaction batch:

PNA alpha_465 in a final concentration (during PNA attachment) of 300 μM PNA beta_594 in a final concentration (during PNA attachment) of 1 μM PNA beta_579 in a final concentration (during PNA attachment) of 1μM PNA beta_554 in a final concentration (during PNA Attachment) of 1 μM These various samples were subjected to the target preparation according to the instructions in the Affymetrix "Expression Analysis Technical Manual" and a GeneChip analysis was carried out on Affymetrix U133A arrays. In contrast to the standard protocol, the following protocol changes were made in the approach using the PNAs: The PNAs were before the Pipette cDNA synthesis together with the T7-Oligo (dT) ₂ primer to the RNA .. Several incubation steps were carried out to attach the primer and the PNAs (10 min 70 ° C; 5 min 45 ° C; 2 min 42 ° C). All further steps were carried out as in the standard protocol.

Table 6: Results of the GeneChip analysis

The signal intensities for the globin mRNA transcripts could be reduced by 40 - 60% by using the PNAs. The proportion of genes which was assessed as "present" on the array could be reduced to the original proportion (Jurkat RNA without in vitro transcripts) in the sample in which the globin was added in vitro transcripts by using the PNA oligonucleotides ,

The signals for the globin mRNAs were not completely suppressed by the use of the PNA oligonucleotides, but the reduction in the globin signal intensities was sufficient to increase the "present call" rate to the original level.

FIG. 3 shows the correlation of the signal intensities of the sample, in which only Jurkat RNA was used, with that of the sample, in which Jurkat RNA was analyzed with added globin in vitro transcripts and using PNA. At this The genes that describe the globin mRNA transcripts have been removed from the analysis.

The correlation coefficient of the signal intensities is 0.9847. This value indicates that the use of the PNAs did not have any non-specific influence on other transcripts represented on the array.

Example 9:

The experiment described in Example 8 was repeated with variation of the PNA oligonucleotide concentrations. For this purpose, the concentration of the oligonucleotide PNA alpha_465 was doubled to 600 nM during the attachment to the globin mRNA.

Table 7: Influence of globin in vitro transcripts through the use of PNA oligonucleotides

Even under these conditions, the negative influence of the globin in vitro transcripts can be reversed by using the PNA oligonucleotides.

Example 10:

total RNA was isolated from HeLa cells. Four samples of this total RNA with a concentration of 2.14 μg / μl were mixed with 42 ng / μl cDNA (generated from the total RNA of the HeLa cells), with a binding buffer from the Superscript ds-cDNA Kit (QIAGEN GmbH, Hilden , DE) and subjected to an RT and a subsequent double strand cDNA synthesis.

After carrying out the enzymatic reactions, the samples were purified using silica spin columns (MinElute Cleanup Kit / QIAGEN GmbH, Hilden, DE). The samples were treated with different washing conditions. Samples 1 and 2 were cleaned according to the manufacturer's cleanup protocol. Samples 3 and 4 were also cleaned primarily in accordance with the manufacturer's Cleanup protocol, but in addition, after application to the silica spin columns or before washing with an ethanol-containing wash buffer, the samples were cleaned in an additional washing step with 700 μl of washing buffer 1 (containing 3.5 M guanidine isothiocyanate, 25 mM sodium citrate, with a pH of 7.0).

After elution of the purified nucleic acids, the proportion of RNA was quantified by RT-PCR analysis (TaqMan analysis / QIAGEN GmbH, Hilden, DE) for p16 RNA (specifically for the detection of RNA) (see FIG. 4).

In addition, the amount of single-stranded cDNA in the eluate was quantified under the different washing conditions (see FIG. 5). A TaqMan PCR system was used to detect p16 cDNA.

The results from FIG. 4 and FIG. 5 clearly show that the additional washing step with the washing buffer according to the invention leads to an extremely efficient depletion of single-stranded nucleic acids (RNA and cDNA).

Example 11:

According to Example 10, 5 μg of genomic double-stranded nucleic acid (dsDNA) and 5 g of single-stranded nucleic acid (RNA) - isolated from HeLa cells - were mixed. After binding to a silica membrane in the presence of a chaotrope and alcohol (MinElute Kit / QIAGEN GmbH, Hilden, DE), the samples were washed under two different conditions before the elution (cleanup): a) washing with washing buffer containing ethanol according to the manufacturer's instructions for the MinElute Kit (QIAGEN GmbH, Hilden, DE) b) Prior washing with 700 μl of washing buffer 1 (3.5 M guanidine isothiocyanate and 25 mM sodium citrate, pH 7.0) before washing with an ethanol-containing washing buffer according to the manufacturer's instructions for the MinElute Kit ( QIAGEN GmbH, Hilden, DE) The samples were analyzed on a denatured formaldehyde agarose gel (before and after the cleanup). The data in FIG. 6 clearly show an efficient depletion of the RNA in the samples which were treated with the washing buffer containing chaotrophic salts in an additional washing step, while the genomic DNA is retained.

Example 12:

As in Example 1, RNA was isolated from whole human blood using the PAXgene Blood RNA Kit (QIAGEN GmbH, Hilden, DE). A target preparation for Affymetrix GeneChip analyzes according to the Affymetrix "Expression Analysis Technical Manual" was carried out with 6μg of the isolated RNA. The cDNA synthesis was primed with an oligo dT-T7 primer. The second strand was then cDNA synthesis. The resulting approaches were followed the binding of the nucleic acids to the silica spin column in two different ways - using the MinElute Cleanup Kit (QIAGEN GmbH, Hilden, DE) - washed or cleaned a) washing on the silica spin column according to the manufacturer's instructions for the MinElute Kit without an additional washing step b ) Washing on the silica spin column including an additional washing step with washing buffer 1 (3.5 M guanidine isothiocyanate and 25 mM sodium citrab pH 7.0) before washing with an ethanol-containing washing buffer according to the manufacturer's instructions for the MinElute Kit.

The purified cDNA was subsequently rewritten into cRNA in an in vitro transcription reaction, and biotinylated nucleotides were incorporated in the process. The samples were purified according to the Affymetrix "Expression Analysis Technical Manual", fragmented and hybridized on a U133A gene chip.

In order to make the results on the different arrays comparable, the average signal intensities of the samples were multiplied by a scaling factor (TGT = 10000). The results of the GeneChip analysis are shown in the following table. Percent scaling factor "present calls" (TGT = 10000) Sample without an additional wash step 34.70 72.37 (standard conditions) Sample with an additional wash step 38.20 54.15 (with wash buffer 1) Table 8: Results of a GeneChip Analysis on U133A gene chips using differently purified target samples.

Due to the additional washing step - and the resulting depletion of single-stranded RNA and cDNA after double-strand synthesis - the proportion of "present calls" on the gene chip increases from 34.7% to 38.2% (by 10%). The scaling factor for the sample without the additional washing step is approx. 33% higher than for the sample that was treated with the additional washing step. This is an indication of an overall higher signal intensity of the gene chip that was hybridized with the sample that was treated with the additional washing step.

Claims

claims

1. Method for the reverse transcription and / or amplification of a product of a reverse transcription of a pool of nucleic acids of a type (A) from a biological sample or an enzymatic reaction, characterized by the selective suppression of the reverse transcription of at least one undesired nucleic acid of the type (A) and / or the selective suppression of the amplification of a product of a reverse transcription of at least one undesired nucleic acid of type (A).

2. The method according to claim 1, characterized in that the nucleic acid of type (A) is mRNA.

3. The method according to claim 1, characterized in that the undesirable nucleic acid of type (A) is an mRNA which has a share of 20% or more in the total mRNA.

4. The method according to any one of claims 2 to 3, comprising the following process steps a) performing a reverse transcription reaction of an RNA from a biological sample or an enzymatic reaction in the presence of at least one oligo-dT primer, b) optionally after step a) performing one cDNA second strand synthesis, c) optionally after step b) purification of the ds-cDNA with simultaneous depletion of all single-stranded nucleic acids from the reaction product from step b), d) optionally after step a) and / or b) and / or c) carry out an amplification the cDNA.

5. The method according to claim 4, characterized in that the process steps a) and / or d) in the presence of at least one molecular species for the selective suppression of the reverse transcription of at least one undesired mRNA, the molecular species preventing the reverse transcription of the undesired mRNA , and / or for the selective suppression of the amplification of a product of the reverse Transcription of at least one undesired mRNA, the molecule species preventing the amplification of the single-stranded or double-stranded cDNA produced from the undesired mRNA.

6. The method according to any one of claims 1 to 5, characterized in that a reverse transcriptase with intrinsic RNase H activity is used in the reverse transcription reaction.

7. The method according to any one of claims 1 to 6, characterized gekennzeichneb that the biological sample is whole blood, muscle tissue or neuronal tissue, or it is a sample contaminated with whole blood, muscle tissue or neuronal tissue.

8. The method according to claim 7, characterized in that the biological sample is whole blood, and that the whole blood is taken up and / or stored in a stabilizing reagent.

9. The method according to claim 8, characterized in that the stabilizing reagent is contained in a blood collection tube and the blood is transferred to the stabilizing reagent immediately upon collection.

10. The method according to claims e or 9, characterized in that the stabilizing reagent contains a tetra-alkyl ammonium salt in the presence of an organic acid.

11. The method according to claim 8 or 9, characterized in that the stabilizing reagent contains at least one guanidine compound, a buffer substance, a reducing agent and a detergent.

12. The method according to any one of claims 1 to 11, characterized gekennzeichneb that the biological sample is whole blood and that the undesirable nucleic acid of type (A) is globin mRNA.

13. The method according to claim 4, characterized gekennzeichneb that for the purification of a ds-cDNA in step c) first of all from step b) and / or from the optional Step d) originating nucleic acids are bound in their entirety to a silica matrix and then the silica matrix is washed with a guanidine-containing washing buffer to deplete the single-stranded nucleic acids.

14. The method according to claim 13, characterized in that one or more silica membrane (s) or silica particles, in particular magnetic silica particles, are used as the silica matrix.

15. The method according to claim 13, characterized gekennzeichneb that the guanidine-containing washing buffer guanidine isothiocyanate and / or guanidine thiocyanate in a concentration of 1 M to 7 M, preferably from 2.5 M to 6 M and particularly preferably from 3 M to 5.7 M. contains.

16. The method according to claim 13, characterized in that the guanidine-containing wash buffer contains guanidine hydrochloride in a concentration of 4 M to 9 M, preferably 5 M to 8 M.

17. The method according to claim 5, characterized in that the molecular species is a DNA oligonucleotide and / or RNA oligonucleotide complementary to the mRNA or to one of the cDNA strands, or a corresponding oligonucleotide from DNA and / or RNA derivatives , or a corresponding DNA and / or RNA oligonucleotide containing modified or artificial nucleotides, quenchers or fluorophores.

18. The method according to claim 17, characterized in that the molecular species has a length of 10 to 60 nucleotides, preferably of 12 to 30 nucleotides.

19. The method according to claim 5, characterized in that the molecular species is a nucleic acid analogue complementary to the mRNA or to one of the cDNA strands.

20. The method according to claim 19, characterized in that the nucleic acid analogue is PNA, LNA or GripNA.

21. The method according to claim 20, characterized in that the PNA has a length of 12 to 20 nucleotide analogs, preferably of 13 to 16 nucleotide analogs.

22. The method according to claim 20, characterized in that the LNA has at least one nucleotide which is a 'locked nucleotide' and that the LNA has a length of 14 to 30 nucleotides, preferably 15 to 22 nucleotides.

23. The method according to claim 20, characterized in that the GripNA has a length of 12 to 30 nucleotide analogs, preferably of 14 to 20 nucleotide analogs.

24. The method according to any one of claims 17 to 23, characterized in that the molecular species binds in the 3 'region of the mRNA or one of the cDNA strands.

25. The method according to any one of claims 5 and 17 to 24, characterized gekennzeichneb that several molecular species are used which are complementary to different regions of one or more specific mRNA (s) or at least one strand of one or more specific cDNA (s) ,

26. The method according to any one of claims 5 and 17 to 24, characterized in that at least one molecular species is used which is complementary to a homologous region of different mRNAs or cDNAs.

27. The method according to any one of claims 5 and 17 to 26, characterized in that the molecular species at its 3 'end has a modification that prevents elongation at the 3' end of the molecular species can be initialized.

28. The method according to claim 5, characterized in that the molecular species is a ribozyme.

29. The method according to claim 28, characterized in that the molecular species is a hammerhead ribozyme or a hairpin ribozyme.

30. The method according to claim 28 or 29, characterized in that the ribozyme consists of RNA or an RNA derivative or embodies fusion products of such ribozymes.

31. The method according to any one of claims 28 to 30, characterized in that the sequence of the ribozymes complementary to the undesired mRNA or cDNA has a length of 12 to 30 nucleotides, preferably of 15 to 25 nucleotides.

32. The method according to claim 5, characterized in that the molecular species is a DNAzyme.

33. The method according to any one of claims 5, 12 and 17, characterized in that the molecular species is a DNA oligonucleotide and the globin mRNA embodies an alpha 1 globin mRNA and / or an alpha 2 globin mRNA, the DNA oligonucleotide a sequence from the group of a) 5 'CTC CAG CTT AAC GGT - phosphate group - 3' b) 5 'TAA CGG TAT TTG GAG - phosphate group - 3' c) 5 'TAA CGG TAT TTG GAG GTC AGC ACG GTG CTC - phosphate group - 3 '.

34. The method according to any one of claims 5, 12 and 17, characterized in that the molecule species is a DNA oligonucleotide and the globin mRNA embodies a beta globin mRNA, the DNA oligonucleotide being a sequence from the group of a) 5 'GTA GTT GGA CTT AGG - phosphate group - 3' b) 5 'ATC CAG ATG CTC AAG - phosphate group - 3' c) 5 'GTA GTT GGA CTT AGG GAA CAA AGG AAC CTT - phosphate group - 3'.

35. The method according to any one of claims 5, 12 and 20, characterized in that the molecular species is a PNA and the globin mRNA embodies an alpha 1 globin mRNA and / or an alpha 2 globin mRNA, the PNA being a Sequence from the group of a) N- CTC CAG CTTAAC GGT-C * b) N- TAA CGG TATTTG GAG -C * c) N- GTC ACC AGC AGG CA -C * d) N- GTG AAC TCG GCG -C * e) N- TGG CAATTC GAC CTC -C * f) N- GAG GTT TAT GGC AAT -C * g) N- ACG GAC GAC CAC TG -C * h) N- GCG GCT CAA GTG -C ^* .

36. The method according to any one of claims 5, 12 and 20, characterized in that the molecule species is a PNA and the globin mRNA embodies a beta globin mRNA, the PNA being a sequence from the group of a) N-GTA GTT GGA CTT AGG -C * b) N- ATC CAG ATG CTC AAG -C ^* c) N- CCC CAG TTT AGT AGT -C ^* d) N- CAG TTT AGT AGT TGG -C ^* e) N- GCC CTT CAT AAT ATC -C ^* f) N- GGA TTC AGG TTG ATG -C * g) N- GAA CTC GAT GAC CTA -C ^* h) N- TGA TGA TTT GAC CCC -C ^* i) N- GGT TGA TGA TTT GAC - C ^* j) N- CTA TAA TAG TTC CCG -C *.

37. The method according to any one of claims 5, 12 and 20, characterized in that the molecular species is an LNA, having at least one nucleotide, which is a 'locked nucleotide', and the globin mRNA is an alpha 1 -globin mRNA and / or an alpha 2-globin mRNA, the LNA being a sequence from the group of a) 5 'CTC CAG CTT AAC GGT - octane diol - 3' b) 5 'TAA CGG TAT TTG GAG - octane diol -3' c ) 5 'GTC ACC AGC AGG CA - octane diol -3' d) 5 'GTG AAC TCG GCG - octane diol -3'.

38. The method according to any one of claims 5, 12 and 20, characterized gekennzeichneb that the molecular species an LNA, comprising at least one nucleotide, which is a 'locked is nucleotide ', and the globin mRNA embodies a beta globin mRNA, the LNA being a sequence from the group of a) 5' GTA GTT GGA CTT AGG - octanediol -3 'b) 5' ATC CAG ATG CTC AAG - Octane diol -3 'c) 5' CCC CAG TTT AGT AGT - octane diol -3 'd) 5' CAG TTT AGT AGT TGG - octane diol -3 'e) 5' GCC CTT CAT AAT ATC - octane diol -3 '.

39. The method according to any one of claims 1, 4 and 5, characterized gekennzeichneb that the amplification comprises an in vitro transcription.

40. The method according to claim 39, characterized in that the in vitro transcription is followed by a DNase digestion and the purification of the cRNA.

41. Use of cRNA resulting from a method according to claim 39 or 40 in a gene expression analysis.

42. Use of cDNA resulting from a method according to one of claims 1 to 38 in a gene expression analysis.

43. Use of a washing buffer containing at least one guanidine compound in a total concentration of 1 M to 9 M, the guanidine compound guanidine isothiocyanate in a concentration of 1 M to 7 M, preferably from 2.5 M to 6 M and particularly preferably is from 3 M to 5.7 M, and / or the guanidine compound is guanidine thiocyanate in a concentration of 1 M to 7 M, preferably from 2.5 M to 6 M and particularly preferably from 3 M to 5.7 M and / or the guanidine compound is guanidine hydrochloride in a concentration of 4 M to 9 M, preferably 5 M to 8 M, in a method for separating single-stranded nucleic acids from double-stranded nucleic acids, preferably in a method for eluting single-stranded nucleic acids from a silica matrix and particularly preferably in a method according to one of claims 1 to 16.

44. Use according to claim 43, wherein the washing buffer further one or more buffer substance (s) in a total concentration of 0 mM to 40 mM and / or one or more additive (s) in a total concentration of 0 mM to 100 mM and / or or more detergent (s) in a total concentration of 0% (v / v) to 20% (v / v), wherein the washing buffer has a pH of 5 to 9, contains.

45. Use according to claim 44, characterized in that the total concentration of the buffer substances is 20 mM to 40 mM.

46. Use according to claim 44 or 45, characterized in that the buffer substance is Tris, Tris-HCl, MOPS, MES, CHES, HEPES, PIPES and / or sodium citrate.

47. Use according to claim 44, characterized in that the additive is a chelating agent.

48. Use according to claim 47, characterized in that the chelating agent is EDTA and / or EGTA.

49. Use according to claim 44, characterized in that the detergent is Tween 20, Triton X 100, Sarcosyl and / or NP 40.

50. Use according to claim 44, characterized in that the pH of the washing buffer is in a range from 6 to 8.

51. Use according to any one of claims 43 to 46, characterized in that the guanidine compound is guanidine isothiocyanate and / or guanidine thiocyanate and the buffer substance is sodium citrate, the washing buffer having a pH in the range from 6 to 8.

52. Use according to claim 51, characterized in that the washing buffer contains guanidine isothiocyanate and / or guanidine thiocyanate in a total concentration of 3.2 M to 3.8 M and sodium citrate in a concentration of 20 mM to 30 mM and a pH of 6 , 8 to 7.7.

3. Use according to claim 52, characterized in that the washing buffer contains guanidine isothiocyanate and / or guanidine thiocyanate in a total concentration of 3.5 M and sodium citrate in a concentration of 25 mM and has a pH of 7.0.