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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

• the invention relates to methods for quantification of the amount of target miRNA in biological samples using normalization.
• the invention relates to methods for quantifying miRNAs in serum, whole blood or platelets samples.
• the invention further relates to kits for quantifying the amount of a target miRNA in a biological sample.
• MicroRNAs are 18 to 25 nucleotides long, noncoding RNAs that regulate gene expression post-transcriptionally by targeting the 3 '-untranslated region of specific mRNAs.
• miRNAs are 18 to 25 nucleotides long, noncoding RNAs that regulate gene expression post-transcriptionally by targeting the 3 '-untranslated region of specific mRNAs.
• several biological processes like cell differentiation and apoptosis, are affected.
• the past decade, many studies have shown the pathophysiological involvement of miRNAs, and several miRNAs were reported as useful biomarkers for specific diseases.
• qPCR quantitative polymerase chain reaction
• RNAs such as RNU6B are often used to normalize, but increasing evidence reveals the regulation of these molecules in pathology. Furthermore, these RNAs are either related to pathology or not stably expressed at a high enough level to serve as a reliable for use in normalization of miRNA qPCR.
• EP2354246A1 discloses a method a method for diagnosing a disease, comprising the steps (a) determining an expression profile of a predetermined set of miRNAs in a biological sample from a patient; and (b) comparing said expression profile to a reference expression profile (from a healthy subject), wherein the comparison of said determined expression profile to said reference expression profile allows for the diagnosis of the disease.
• the reference expression profile is the expression profile of the same set of miRNAs in a biological sample originating from the same source as the biological sample from a patient but obtained from a healthy subject.
• the disadvantage of using the same miRNA as a reference control is that there is no correction for inter sample variation.
• the inventors propose a novel, more standardized method for the normalization of RT-qPCR experiments on circulating miRNAs.
• the invention is based on the surprising finding of a miRNA panel comprising miRNAs that are stably expressed in the circulation of healthy individuals and which are not related to a disease. These miRNA may therefore be used as normalization panel which can be universally used for miRNA qPCR experiments on circulating miRNAs.
• Candidate miRNAs with a stable expression were selected from miRNA microarray experiments from the GEO database of either whole blood, isolated platelet' s or serum array experiments. For each sample type the inventors selected those miRNAs that were least variable and sufficiently highly expressed in available array experiments, performed on at least two different platforms. The stability of these candidate normalization miRNAs was further assessed using the geNorm and Normfinder algorithms in a qPCR cohort of 10 patients with coronary artery disease and 10 healthy controls, resulting in a suitable normalization panel. The inventors constructed normalization panels for the normalization of miRNA qPCR experiments which are specifically suitable for use in whole blood, isolated platelets and serum samples.
• the inventors confirmed that the performance of the whole blood normalization panel is superior to other frequently used normalization methods on precision and reproducibility measures.
• the inventors further show that the use of RNU6B for the normalization of qPCR experiments on circulating miRNAs is not feasible, since RNU6B is not reliably detectable in the circulation.
• Mir- 16 which was apart from our normalisation panel the best normalisation candidate, showed les precision and less accuracy as normalisation miRNA as compared to the different normalisation panels.
• the inventors selected data on healthy controls from multiple microarray experiments studying a variety of diseases. Exclusion of candidate miRNAs that were reported to be regulated in any disease and the use of the geNorm and Normfinder algorithms resulted in a panel of stably expressed normalization miRNAs. Thus, the combination of including different miRNA micro-array experiments in the selection procedure and the use of two independent algorithms to select the most stable normalization miRNAs has led to the selection of reliable normalization panels for circulating miRNAs that can universally be used.
• the invention provides a method of quantifying the amount of a target microRNA (miRNA) in a biological sample, the method comprising: determining the amount of the target miRNA in the biological sample; determining the amount of at least a first reference miRNA selected from the group consisting of miR-1280 , miR-1260a miR-718, miR-484, MiR-130b-3p, miR-342-3p miR-151-3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p and miR-18a-5p, in the biological sample; and normalizing the target miRNA measurement based on the amount of at least said first reference miRNA.
• said first reference miRNA and said target miRNA are not the same.
• said group consists of miR-130b-3p, miR-342-3p, miR-1280, miR- 1260a, miR- 718, miR-151a-3p, miR-331-3p, miR-29c-3p, miR-148b-3p and miR-18a-5p.
• said method further comprises measuring a second reference miRNA of said group in the biological sample and normalizing the target miRNA levels to the first miRNA and the second reference miRNA.
• said second reference miRNA and said target miRNA are not the same.
• said method further comprises amplifying the target miRNA and the at least one reference miRNA in the reaction volume. Said amplification preferably includes realtime polymerase chain reaction amplification.
• said first and second reference miRNA is selected from the group consisting of miR-130b-3p and miR-342-3p.
• said first and second reference miRNA is selected from the group consisting of miR-1280, miR-1260a miR-718, miR-484, more preferably miR-1280 and miR-1260a.
• An advantage of these miRNAs is that they are very stable in biological samples of serum.
• said first and second reference miRNA are said first and second reference miRNA is selected from the group consisting of miR-151a-3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p and miR-18a-5p, more preferably miR-148b-3p and miR-18a-5p.
• An advantage thereof is that these miRNAs are very stable in biological samples comprising isolated platelets.
• a combination of 6 miRNAs (miR-151a-3p, miR-28-5p, miR-331-3p, miR- 29c-3p, miR-148b-3p and miR-18a-5p) is used for normalization, as this combination has the lowest V value.
• said biological sample comprises serum, whole blood or platelets.
• the invention further provides a kit for quantifying the amount of a target miRNA in a biological sample comprising an amplification primer set, comprising at least one primer comprising a sequence that is complementary to a portion of said first reference miRNA as defined above.
• said amplification primer set further comprises a sequence that is complementary to a portion of said second reference miRNA as defined above.
• the kit of the invention further comprises a second amplification primer set, wherein at least one primer comprises a sequence that is complementary to a portion of a target miRNA.
• the kit according to the invention further comprises a first probe comprising a sequence that is complementary to a portion of the target miRNA and a second probe comprising a sequence that is complementary to a portion of the reference miRNA, wherein the first and second probes are distinguishably detectable.
• Figure 1 (A) shows the ranking of candidate normalization miRNAs according to average expression stability. In a stepwise manner, the least stable miRNAs with the highest M values were excluded until miR-130b-3p and miR-342-3p remained.
• Figure 1(B) shows the determination of the optimal number of normalization miRNAs.
• the optimal normalization panel consists of the number of miRNAs with the lowest V value. In this case the optimal V value is achieved when using 2 normalization miRNAs.
• Figure 2 shows the precision of two normalisation methods, either normalisation with MIR- 16 or with the normalisation panel.
• Figure 2 (A) shows the correlation between two identical qPCR
• FIG. 1 shows the correlation between two identical qPCR measurements of miR-494 measured in whole blood on the same sample and normalised for miR-16. This shows worse results with a correlation of 0.27.
• Figure 3 shows the accuracy analysis of the isolated platelet normalization panel. Previous miRNA microarray experiments showed that variable X is positively correlated with miR-A expression.
• A Using qPCR without any normalization method the inventors were not able to confirm these data.
• B When qPCR data was normalized for the whole blood normalization panel the correlation between variable X and miR-A expression could be confirmed.
• miRNA includes human miRNAs, mature single stranded miRNAs, precursor miRNAs, and variants thereof, which may be naturally occurring or synthetic. Synthetic or naturally occurring miRNAs may be modified to include chemical groups other than hydroxy or phosphate at their 5' termini, sugar, and/or base modifications. In some instances the term “miRNA” also includes primary miRNA transcripts and duplex miRNAs. The term includes target miRNAs, miRNAs, and reference miRNAs (see below). The term “mature,” when modifying miRNA or a specific miRNA, refers to the mature sequence(s) processed from the corresponding pre- miRNA sequence that are present in a biological sample.
• miRNAs including human mature and precursor sequences
• sequences for particular miRNAs are reported in the miRBase: Sequences Database (http:/microrna.sanger.ac.uk; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144; Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111).
• the skilled artisan will appreciate that scientific consensus regarding the precise nucleic acid sequence for a given miRNA, in particular for mature forms of the miRNAs, may change with time.
• MiRNAs detected by assays of this application include naturally occurring sequences for the miRNAs.
• miR-1280, miR-1260a, miR-718, miR-484, MiR-130b-3p, miR-342-3p miR-151- 3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p, miR-18a-5p and so on as used herein refer to the miRNAs as retrieved in miRBase version 21. Exemplary sequences of the miRNAs are listed in Table 1.
• miR- ⁇ 151-3p CUAGACUGAAGCUCCUUGAGG
• miR- ⁇ 28-5p AAGGAGCUCACAGUCUAUUGAG
• miR- ⁇ 331-3p GCCCCUGGGCCUAUCCUAGAA
• miR- ⁇ 29c UAGCACCAUUUGAAAUCGGUUA 15. miR-1225-3p: UGAGCCCCUGUGCCGCCCCCAG
• miR-718 CUUCCGCCCCGCCGGGCGUCG
• miR-484 UCAGGCUCAGUCCCCUCCCGAU
• miR-342-3p UCUCACACAGAAAUCGCACCCGU
• target miRNA refers to any miRNA of interest.
• a target miRNA or reference miRNA is preferably amplified prior to or during quantification. In other embodiments, the miRNA is not amplified as part of the quantification process.
• Amplification Reactions Many methods exist for amplifying miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs. Suitable nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. In certain preferred embodiments, more than one amplification method is used, such as reverse transcription followed by real time PCR (Chen et al., Nucleic Acids Research, 33(20):el79 (2005)).
• a typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species.
• a typical PCR reaction includes three steps: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
• Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation.
• the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps.
• a reverse transcription reaction (which produces a complementary cDNA sequence) is performed prior to PCR reactions.
• Reverse transcription reactions include the use of, e.g., a RNA -based DNA polymerase (reverse transcriptase) and a primer.
• a set of primers is used for each target sequence.
• the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified. In preferred
• a primer is about 15 to about 35 nucleotides in length. In other preferred embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. In additional preferred embodiments, a primer is at least 35 nucleotides in length.
• a forward primer can comprise at least one sequence that anneals to a target miRNA and alternatively can comprise an additional 5' non- complementary region.
• a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA.
• the reverse primer may be independent of the target miRNA or reference miRNA sequence, and multiple target miRNAs or reference miRNAs may be amplified using the same reverse primer.
• a reverse primer may be specific for a target miRNA.
• two or more miRNAs are amplified in a single reaction volume (one or more target miRNAs and one, two, three, or more reference miRNAs, for example). Normalization may alternatively be performed in separate reaction volumes.
• One preferred embodiment includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least one miRNA of interest and at least one reference miRNA in one reaction volume by using more than one pair of primers and/or more than one probe.
• the primer pairs may comprise at least one amplification primer that uniquely binds each miRNA, and the probes are preferably labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.
• Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
• the qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA -based thermostable DNA polymerase.
• a "hot start" approach may be used to maximize assay performance (U.S. Pat. Nos. 5,411 ,876 and 5,985,619).
• the components for a reverse transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Pat. Nos. 5,550,044, 5,413,924, and 6,403,341).
• labels, dyes, or labeled probes and/or primers are used to detect amplified or unamplified miRNAs.
• amplification may or may not be required prior to detection.
• One skilled in the art will recognize the detection methods where miRNA amplification is preferred.
• a probe or primer may include Watson-Crick bases or modified bases.
• Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Pat. Nos. 5,432,272, 5,965,364, and 6,001,983.
• bases are joined by a natural phosphodiester bond or a different chemical linkage.
• Different chemical linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Pat. No. 7,060,809.
• oligonucleotide probes or primers present in a multiplex amplification are suitable for monitoring the amount of amplification product produced as a function of time.
• probes having different single stranded versus double stranded character are used to detect the nucleic acid.
• Probes include, but are not limited to, the 5'- exonuclease assay (e.g., TaqMan(TM)) probes (see U.S. Pat. No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Pat. Nos.
• stemless or linear beacons see, e.g., WO 9921881, U.S. Pat. Nos. 6,485,901 and 6,649,349
• PNA peptide nucleic acid
• Molecular Beacons see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091
• linear PNA beacons see, e.g. U.S. Pat. No.
• Sunrise(TM)/AmplifluorB(TM)probes see, e.g., U.S. Pat. No. 6,548,250
• stem-loop and duplex Scorpion(TM) probes see, e.g., U.S. Pat. No. 6,589,743
• bulge loop probes see, e.g., U.S. Pat. No. 6,590,091
• pseudo knot probes see, e.g., U.S. Pat. No. 6,548,250
• cyclicons see, e.g., U.S. Pat. No. 6,383,752
• MGB Eclipse(TM) probe Epoch Biosciences
• hairpin probes see, e.g., U.S. Pat.
• one or more of the primers in an amplification reaction can include a label.
• different probes or primers comprise detectable labels that are distinguishable from one another.
• a nucleic acid, such as the probe or primer may be labeled with two or more distinguishable labels.
• a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody-antigen, ionic complexes, hapten-ligand (e.g., biotin-avidin).
• use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
• MiRNAs can be detected by direct or indirect methods.
• a direct detection method one or more miRNAs are detected by a detectable label that is linked to a nucleic acid molecule.
• the miRNAs may be labeled prior to binding to the probe. Therefore, binding is detected by screening for the labeled miRNA that is bound to the probe.
• the probe is optionally linked to a bead in the reaction volume.
• nucleic acids are detected by direct binding with a labeled probe, and the probe is subsequently detected.
• the nucleic acids such as amplified miRNAs, are detected using FlexMAP Microspheres (Luminex) conjugated with probes to capture the desired nucleic acids.
• FlexMAP Microspheres Luminex
• Some methods may involve detection with polynucleotide probes modified with fluorescent labels or branched DNA (bDNA) detection, for example.
• nucleic acids are detected by indirect detection methods.
• a biotinylated probe is combined with a streptavidin- conjugated dye to detect the bound nucleic acid.
• the streptavidin molecule binds a biotin label on amplified miRNA, and the bound miRNA is detected by detecting the dye molecule attached to the streptavidin molecule.
• the streptavidin-conjugated dye molecule comprises
• Labels include, but are not limited to: light-emitting, light-scattering, and light-absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L., Nonisotopic DNA Probe Techniquies, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997).).
• Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g., U.S. Pat. Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamines (see, e.g., U.S. Pat. Nos.
• fluorescein dyes include, but are not limited to, 6- carboxyfluorescein; 2',4',1,4,-tetrachlorofluorescein; and 2',4',5',7',1,4-hexachlorofluorescein.
• the fluorescent label is selected from SYBR-Green, 6- carboxyfluorescein ("FAM”), TET, ROX, VIC(TM), and JOE.
• FAM 6- carboxyfluorescein
• TET 6- carboxyfluorescein
• ROX ROX
• VIC(TM) VIC(TM)
• JOE JOE
• labels are different fluorophores capable of emitting light at different, spectrally- resolvable wavelengths (e.g., 4-differently colored fluorophores); certain such labeled probes are known in the art and described above, and in U.S. Pat. No.
• a dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some preferred embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
• labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
• intercalators and intercalating dyes including, but not limited to, ethidium bromide and SYBR-Green
• minor-groove binders include, but not limited to, ethidium bromide and SYBR-Green
• cross-linking functional groups see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
• methods relying on hybridization and/or ligation to quantify miRNAs may be used, including oligonucleotide ligation (OLA) methods and methods that allow a distinguishable probe that hybridizes to the target nucleic acid sequence to be separated from an unbound probe.
• OLA oligonucleotide ligation
• HARP-like probes as disclosed in U.S. Publication No.
• 2006/0078894 may be used to measure the quantity of miRNAs.
• the probe after hybridization between a probe and the targeted nucleic acid, the probe is modified to distinguish the hybridized probe from the unhybridized probe. Thereafter, the probe may be amplified and/or detected.
• a probe inactivation region comprises a subset of nucleotides within the target hybridization region of the probe.
• a post-hybridization probe inactivation step is carried out using an agent which is able to distinguish between a HARP probe that is hybridized to its targeted nucleic acid sequence and the corresponding the unhybridized HARP probe.
• the agent is able to inactivate or modify unhybridized HARP probe such that it cannot be amplified.
• a probe ligation reaction may be used to quantify miRNAs.
• MLPA Multiplex Ligation-dependent Probe Amplification
• MLPA probes have flanking PCR primer binding sites. MLPA probes can only be amplified if they have been ligated, thus allowing for detection and quantification of target miRNA or reference miRNA.
• Normalization Methods of normalization and kits for normalizing miRNA detection assays are provided herein. The methods correct for sample-to-sample variability by comparing a target measurement in a sample to one or more internal controls. Normalization of miRNA quantification assays reduces systematic (non-biological) and non-systematic differences between samples, and is critical for accurate measurement of differential miRNA expression, for example.
• Biological reasons may include variabilities in tissue procurement or storage, inconsistencies in RNA extraction or quantification, or differences in the efficiency of the reverse transcription and/or PCR steps.
• Biological reasons may include sample-to-sample heterogeneity in cellular populations, differences in bulk transcriptional activity, or alterations in specific miRNA expression that is linked to an aberrant biological program (e.g., a disease state).
• results from qRT-PCR assays should be normalized against a relevant endogenous target or targets to minimize controllable variation, and permit definitive interpretations of nominal differences in miRNA expression.
• Preferred embodiments comprise multiplex methods for quantifying and normalizing the amount of target miRNA in a biological sample.
• the amount of one or more target miRNAs is measured in a reaction volume, and the amount of at least said first reference miRNA measured in the reaction volume.
• the amount of target miRNA is normalized based on the amount of at least said first reference miRNA.
• two or three reference further miRNAs are measured.
• further reference miRNAs are measured.
• the one or more target miRNA measurements are normalized to the measurement of two, three, four, or more reference miRNAs.
• Luminex technology allows for detection of as many as 100 unique analytes in one sample.
• the FlexMir assay includes 4 snoRNAs as controls for signal normalization.
• the relative expression of a target miRNA in two or more biological samples can be quantified and normalized to the amount of a reference miRNA.
• the data are normalized to the measured quantity of said one reference miRNA.
• a mean of the normalizers e.g. arithmetic mean or geometric mean
• the threshold cycle (Ct) values obtained from q-PCR experiments may be normalized to the geometric mean of two or more normalizers.
• Data represented on a linear scale absolute expression data
• expression levels may be normalized using a comparative Ct method for relative quantification between samples or sample types.
• the general methods for conducting such assays are described, e.g., in Real-Time PCR Systems: Applied Biosystems 7900HT Fast Real-Time PCR System, and 7300/7500 Real-Time PCR Systems, Chemistry Guide, Applied Biosystems, 2005, Part No. 4348358.
• Preferred embodiments of the invention include measuring the amount of at least one reference miRNA, and normalizing the amount of a target miRNA to the amount of at least one miRNA(s). Further normalizers suitable for use in the claimed methods are stably expressed and do not show significant differential expression in healthy or in diseased individuals.
• said normalizers are identified using the NormFinder (Andersen et al., Cancer Res. 64 (15):5245-5250 (2004)) or geNorm (Vandesompele et. al., Genome Biol. 3(7): research 0034.1 -0034.11 (2002)) algorithms based on various qRT-PCR data from human cell and tissue collections. Additional statistical methods are known in the art for identifying stably expressed members of a group, and are also contemplated for use to identify miRNA normalizers.
• normalizers are identified by using the NormFinder or geNorm algorithms to analyze data from normal and disease tissue samples. There are many suitable reference samples that can be used to identify reference miRNAs.
• normalizers are identified using both NormFinder and geNorm.
• Preferred embodiments include measuring the amount of a target miRNA and a reference miRNA, and normalizing the target miRNA level to the reference miRNAs. Additional preferred embodiments include measuring the amount of a first and a second reference miRNA, and normalizing the target miRNA level to the first and second miRNAs. Further preferred embodiments include quantifying the relative expression of target miRNAs between biological samples by (a) measuring the amount of a target miRNA and a first reference miRNA in a first biological sample, (b) measuring the amount of a target miRNA sequence and the first reference miRNA in a second biological sample, and (c) normalizing the target miRNA level to the reference miRNA level for the first and second sample.
• the one or more reference miRNA(s) is/are chosen from miR-
• the one or more reference miRNA(s) is/are chosen from miR- miR-1280, miR- 1260a miR-718 and miR-484. These miRNAs have been found to be highly suitable for normalization of miRNAs in serum samples. In a highly preferred embodiment, the one or more reference miRNA(s) is/are chosen from miR- miR-1280 and miR-1260a, as this combination has an excellent stability value.
• the one or more reference miRNA(s) is/are chosen from MiR-130b-3p, miR-342-3p. These miRNAs have been found to be highly suitable for normalization of miRNAs in whole blood samples.
• the one or more reference miRNA(s) is/are chosen from miR- miR-151a-3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p and miR-18a-5p. These miRNAs have been found to be highly suitable for normalization of miRNAs in platelets samples.
• the one or more reference miRNA(s) is/are chosen from , miR-148b-3p and miR-18a-5p, as this combination has an excellent stability value.
• miRNAs for normalization. In other preferred embodiments, not more than 5, 4, 3 or 2 miRNAs.
• the amount of target miRNA in a biological sample is normalized to the amount of at least one reference miRNA in the biological sample.
• a “biological sample” is any sample or specimen derived from a human.
• the biological sample may be a patient sample.
• a “patient sample” is any biological specimen from a patient.
• the term includes, but is not limited to, biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid, lavage fluid, semen, and other liquid samples, as well as cells and tissues of biological origin.
• the term also includes cells isolated from a human or cells derived therefrom, including cells in culture, cell supernatants, and cell lysates.
• tissue biopsy samples tissue biopsy samples, tumor biopsy samples, stool samples, and fluids extracted from physiological tissues, as well as cells dissociated from solid tissues, tissue sections, and cell lysates.
• a biological sample may be obtained or derived from tissue types including but not limited to lung, liver, placenta, bladder, brain, heart, colon, thymus, ovary, adipose, stomach, prostate, uterus, skin, muscle, cartilage, breast, spleen, pancreas, kidney, eye, bone, intestine, esophagus, lymph nodes and glands.
• biological sample encompasses samples that have been manipulated in any way after their procurement, such as by treatment with preservatives, cellular disruption agents (e.g. lysing agents), solubilization, purification, or enrichment for certain components, such as polynucleotides, in certain aspects. Also, derivatives and fractions of patient samples are included.
• a sample may be obtained or derived from a patient having, suspected of having, or recovering from a disease or pathological condition.
• Diseases and pathological conditions include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.
• Diseases e.g. cancers
• said biological sample comprises serum, whole blood or platelets
• kits of reagents and macromolecules for carrying out the normalization assays provided herein.
• the invention provides a kit for quantifying a target miRNA sequence and a reference miRNA sequence in a reaction volume.
• the kits include nucleic acid sequences that are identical or complementary to a portion of at least one target miRNA and at least one reference miRNA as defined above, for the detection of the target miRNA and the reference miRNA.
• the kits comprise at least one primer for the detection of a reference miRNA and a target miRNA.
• the kits comprise at least one probe specific to a reference miRNA and a target miRNA. The sequence-specific primers or probes are distinguishably labeled, allowing detection of at least one reference miRNA and at least one target miRNA in a single reaction volume.
• kits further optionally comprise an enzyme for carrying out the method described herein, including but not limited to a polymerase such as a reverse transcriptase or a DNA polymerase, or a ligase.
• a polymerase such as a reverse transcriptase or a DNA polymerase, or a ligase.
• the kits preferably include nucleic acid molecules that are identical or complementary to a target miRNA and/or a reference miRNA. Such molecules may serve as absolute standards for creating standard curves to quantify the unknown levels of target in the sample of interest.
• kits preferably comprises multiple amplification primer sets, wherein at least one of the primers in each of the primer sets comprises a sequence that is
• kits complementary to a portion of at least two miRNAs, such as a target miRNA and a reference miRNA, or two reference miRNAs, for example.
• the kits preferably further comprise at least two probes complementary to a portion of at least two miRNAs.
• the kit preferably also comprises reagents for reverse transcribing RNA to a DNA template and/or reagents, including primers, for amplification of the target DNA.
• Such a kit preferably includes one or more buffers, such as a reaction, amplification, and/or a transcription buffer, compounds for preparing a RNA sample, for preparing a DNA sample, and components for isolating and/or detecting an amplification product, such as a probe or label, for example.
• kits of the invention preferably include one or more of the following (consistent with methods, reagents, and compositions discussed above): components for sample purification, including a lysis buffer with a chaotropic agent; a glass-fiber filter or column; an elution buffer; a wash buffer; an alcohol solution; and a nuclease inhibitor.
• the components of the kits may be packaged either in aqueous media or in lyophilized form, for example, and will be provided in a suitable container.
• the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
• the container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted.
• the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
• kits of the present invention will also typically include a means for containing the RNA, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
• the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
• kits may also include components that preserve or maintain DNA or RNA, such as reagents that protect against nucleic acid degradation.
• Such components may be nuclease or RNase-free or protect against RNases, for example. Any of the compositions or reagents described herein may be components in a kit.
• reagents in a kit for reverse transcription and q-PCR of a target miRNA and a reference miRNA include reverse transcriptase, a reverse transcriptase primer, corresponding PCR primer sets, a thermostable DNA polymerase, and two distinguishable detection reagents which may include scorpion probes, probes for a fluorescent 5' nuclease assay, molecular beacon probes, single dye primers or fluorescent dyes specific to double-stranded DNA (e.g. ethidium bromide).
• the kit may also include multiple reverse transcriptase primers to one or more additional miRNAs, such as a target miRNA and/or a second reference miRNA.
• Additional materials may include a suitable reaction container, a barrier composition, reaction mixtures for reverse transcriptase and PCR stages (including buffers and reagents such as dNTPs), nuclease- or RNAse-free water, RNase inhibitor, and/or any additional buffers, compounds, co-factors, ionic constituents, proteins, enzymes, polymers, and the like that may be used in reverse transcriptase and/or PCR stages of the reactions.
• reaction mixtures for reverse transcriptase and PCR stages including buffers and reagents such as dNTPs
• nuclease- or RNAse-free water including RNase inhibitor, and/or any additional buffers, compounds, co-factors, ionic constituents, proteins, enzymes, polymers, and the like that may be used in reverse transcriptase and/or PCR stages of the reactions.
• Candidate miRNAs were selected from previously performed studies that reported miRNA microarray data in the Gene Expression Omnibus (GEO). PubMed en GEO were extensively searched for studies in which miRNA microarrays were performed on whole blood, platelet, or serum samples. Microarray experiments were included if they reported individual miRNA expression of at least ten healthy controls. The inventors excluded two colour arrays, array experiments that reported ratio or Z transformed data, arrays on data of pooled individuals and arrays of which normalized data was not available. Furthermore, experiments with low miRNA expression levels were also excluded.
• GEO Gene Expression Omnibus
• Normfinder identifies the optimal normalisation miRNA by ranking all candidate miRNAs in terms of stability in a given sample set (Andersen et al. 2004). Additionally, it identifies the optimal pair of miRNAs that can be used for normalisation. GeNorm uses a stability measure that quantifies to what extent the expression ratio of two candidate miRNAs is identical in all samples.
• the algorithm determines the pairwise variation with all other candidates and determines the stability measure M (Vandesompele et al. 2002 Genome Biol [Internet]. 2002;3:RESEARCH00347).
• GeNorm also determines a V-value, that is the pairwise variation between two consecutive normalisation miRNAs starting with the candidate miRNA with the lowest M value.
• the combination of miRNAs that resulted in the lowest V-value was selected as the optimal set of normalisation miRNAs (Mestdagh et al. Genome Biol [Internet]. 2009 [cited 2013 Mar 6] ;10:R64.).
• the final normalisation panels consisted of those candidate miRNAs selected by both geNorm and Normfinder. If both algorithms selected different normalisation panels, both panels were combined in the final normalisation panel. Validation of the whole blood normalization panel
• the accuracy of the normalization panel was analysed in isolated platelets of 25 healthy volunteers, in which the inventors previously determined the expression level of a target miRNA on micro array. Accuracy of the normalization panel was analysed by comparing the PCR results of the expression of miR-A with the array results, according to the different normalisation methods, either the platelet normalisation panel, RNU6B or miR-16. The normalisation method which best reproduced the array results of this study was supposed to be the best normalisation method.
• MiR-130b-3p and miR-342-3p were selected as the best combination of genes with a stability value of 0.085.
• the geNorm analysis confirmed that miR-130b-3p was the most stable (figure la). No combination of reference miRNAs reached a V-value ⁇ 0.15 (figure lb). Therefore, the inventors selected the combination of miRNAs with the lowest V-value.
• This panel consisted of miR-130b-3p and miR-342-3p and had a V-value of 0.20. Since this panel corresponds with the Normfinder results, the inventors consider it a sufficient reference panel. Adding more candidate miRNAs did not improve the results. Platelets
• Normfinder selected miR-148b-3p as the most stable reference miRNA with a stability value of 0.086.
• MiR-148b-3p and miR-18a-5p were selected as the best combination of genes with a stability value of 0.064.
• GeNorm analysis revealed that miR-374b was the least stable reference miRNA, whereas miR- 151a-3p was most stable (supplementary figure 1).
• miR-151a-3p, miR-28-5p, miR-331-3p, miR-29c-3p, miR-148b-3p and miR-18a-5p the lowest V value was calculated. This included the miRNAs selected by Normfinder.
• geNorm showed that miR-1238 was the least stable candidate miRNA, whereas miR-1260a was the most stable candidate miRNA.
• a combination of 4 candidate miRNAs reached a V-value ⁇ 0.15. This panel consisted of miR- 1260a, miR-1280, miR-718 and miR- 484, and corresponded with the combination of candidate miRNAs selected by Normfinder.
• the performance of the platelet normalization panel was analysed as a proof of principle by assessing the precision and accuracy.

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