Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

• 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.
• nucleic acids 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.
• 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.
• 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.
• RNA deoxyribonucleic acids
• DNA deoxyribonucleic acids
• RNA ribonucleic acids
• copy DNA cDNA
• genomic DNA gDNA
• messenger RNA mRNA
• transfer RNA tRNA
• rRNA ribosomal RNA
• small nuclear RNA snRNA
• pDNA plasmid DNA
• viral DNA viral RNA etc.
• modified or artificial nucleic acids or also nucleic acid analogues such as peptides nucleic acids (PNA) or Locked Nucleic Acids (LNA) etc.
• PNA peptides nucleic acids
• LNA Locked Nucleic Acids
• RT-PCR reverse transcription reactions with polymerase chain reaction
• 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.
• RNA transcripts are also reversely transcribed.
• 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.
• 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.
• RNA level 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).
• IVT in vitro transcription
• 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.
• RNA from a sample that contains both types of nucleic acid includes digestion with RNase.
• 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.
• the RNase can only be partially removed completely from the sample.
• 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.
• 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).
• the nucleic acid of type (A) is mRNAs, which may be natural mRNAs or those derived from in vitro transcription reactions.
• 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.
• 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.
• 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.
• 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 intents GmbH, Stuttgart, DE) can be used.
• WO 01/060517 / Antigen principless 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).
• the individual method steps can also be designed differently.
• 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.
• 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).
• an amplification of the cDNA can be carried out.
• 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).
• 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).
• 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.
• 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 ,
• a cDNA second-strand synthesis can then optionally be carried out using a method known per se, including the common reagents.
• 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.
• a specific reverse transcriptase e.g. LabelStar RT / QIAGEN GmbH, Hilden, DE
• a specific reverse transcriptase 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).
• 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).
• ss cDNA e.g. ss cDNA, viral cDNA etc.
• 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.
• the various unlabeled nucleic acids in solution compete for the binding with the labeled cRNA transcripts to the probes on the array
• 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.
• 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).
• 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).
• 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.
• 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.
• all non-hybridized, and thus still single-stranded, transcripts are separated from the nucleic acid mixture.
• 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.
• 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) .
• 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.
• guanidine hydrochloride can also be used according to the invention, with a concentration of 4 M to 9 M, preferably 5 to 8 M.
• 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
• 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.
• common buffer substances such as Tris, Tris-HCl, MOPS, MES, CHES, for example
• wash buffer composition can be added.
• chelating agents for example EDTA, EGTA or other suitable compounds
• detergents for example Tween 20, Triton X 100, Sarcosyl, NP40 etc.
• 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
• Guanidine thiocyanates can be used in combination with or instead of guanidine isothiocyanates.
• 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).
• 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.
• 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.).
• 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.
• 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.
• 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.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• NASBA nucleic acid sequence-based amplification
• 3SR self-sustained sequence replication
• 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.
• 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.
• 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, Rothstadt, 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.
• 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.
• molecule species which is used for sequence-specific blocking 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.
• the labeling of RNA is prevented by hybridizing the RNA with tightly binding molecules.
• RNAzymes 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.
• 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.
• 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 '
• 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 '
• 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 *
• 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.
• the molecular species is an LNA which has at least one nucleotide which is a locked nucleotide 1
• the globin mRNA is an alpha 1 globin mRNA and / or an alpha 2 globin mRNA
• 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 '
• 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
• 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 '
• 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.
• RNA preparations which have a high proportion of overrepresented transcripts e.g. globin mRNA transcripts
• 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.
• 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.
• Band L RNA size standard, band 1: generated cRNA at a final PNA concentration of 10 ⁇ M, band 2: generated cRNA at a final PNA concentration of 1.0 ⁇ M, band 3: generated cRNA at a final PNA concentration of 0, 1 ⁇ M, band 4: generated cRNA at a final PNA concentration of 0.01 ⁇ M, band 5: generated cRNA at a final PNA concentration of 0.001 ⁇ M band 11: generated cRNA without addition of PNAs (comparative sample).
• 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.
• 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.
• 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).
• Table 1 Results of a GeneChip analysis on U133A GeneChips using different reverse transcriptases.
• 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.
• 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.
• 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.
• RNA of a blood donor was isolated as in Example 1 using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH).
• PAXgene Blood RNA System PreAnalytix, Hombrechticon, CH.
• 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:
• RNA of a blood donor was isolated as in Example 1 using the PAXgene Blood RNA System (PreAnalytix, Hombrechticon, CH).
• PAXgene Blood RNA System PreAnalytix, Hombrechticon, CH.
• PNA sequences PE Biosystems
• alpha_465 N- TAA CGG TAT TTG GAG -C *
• beta_554 N- GTA GTT GGA CTT AGG -C *
• RNA 5 ⁇ g 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.
• 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.
• the PNAs were added in a final concentration of 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1, 0 ⁇ M and 10 ⁇ M. 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.
• FIGS. 1 and 2 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.
• 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 ⁇ g 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).
• PNAs sequences see below
• PNAs were added at a final concentration of 0.5 ⁇ M, 1.0 ⁇ M 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.
• 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:
• 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 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 ⁇ M, the transcript level for alpha globin remains unaffected.
• PAXgene Blood RNA System PreAnalytix, Hombrechticon, CH.
• the target samples for the RNA samples of both donors were prepared using the following protocol variants:
• 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 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%.
• 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
• T7-oligo (dT) 2 primer Three different approaches were compared:
• Jurkat RNA 2.
• Jurkat RNA with spiked globin in vitro transcripts 3.
• PNAs peptide nucleic acids
• 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.
• 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 8 The experiment described in Example 8 was repeated with variation of the PNA oligonucleotide concentrations.
• concentration of the oligonucleotide PNA alpha_465 was doubled to 600 nM during the attachment to the globin mRNA.
• 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 ⁇ l of washing buffer 1 (containing 3.5 M guanidine isothiocyanate, 25 mM sodium citrate, with a pH of 7.0).
• washing buffer 1 containing 3.5 M guanidine isothiocyanate, 25 mM sodium citrate, with a pH of 7.0.
• 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).
• 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.
• Example 10 5 ⁇ g 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 ⁇ l 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.
• 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 6 ⁇ g 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.
• washing buffer 1 3.5 M guanidine isothiocyanate and 25 mM sodium citrab pH 7.0
• 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.
• 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.

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