miRNA profiling in serum & plasma

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Profiling in serum & plasma: …

Profiling in serum & plasma:
On the road to biomarker development.

The slides describe the workflow for circulating miRNA profiling from the sample isolation and preparation through to data analysis & normalization.

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  • 1. Isolation Quantification Functionalization The miRNA Revolution miRNA Profiling in serum & plasma: On the road to biomarker development Eric Lader, PhD Senior Director, R&D eric.lader@qiagen.com -1- Sample & Assay Technologies
  • 2. RNA interference: A natural phenomenonDiscovery tool, potential therapeutic May 26, 2003 -2- Sample & Assay Technologies
  • 3. Canonical pathway of microRNA biogenesis microRNA Gene DNA Transcribed by RNA Polymerase II as NUCLEUS POL II a long primary transcript (pri-miRNAs), Pri-miRNA which may contain more than one miRNA. Drosha-DGCR8 In the nucleus, Pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III- Pre-miRNA like enzyme Drosha CYTOPLASM Exportin Pre-miRNAs are then exported to the Exportin Cytosol by Exportin 5 DICER-TRBP In the cytosol RNAse III-like Dicer, processes these precursors to mature mature miRNA miRNAs Ago RISC Assembly These miRNAs are incorporated in RISC RISCHigh homology Partial homology miRNAs with imperfect base pairing to the target mRNA, lead to translational repression and/or mRNA degradation mRNA cleavage Translational Repression mRNA degradationKrol, J et.al., (2010) Nature Rev Genetics, 11, 597; Winter, J. et.al., (2009) Nature Cell Biology, 11, 228 -3- Sample & Assay Technologies
  • 4. Potential events leading to disruption of ‘normal’ miRNA:target interaction in diseaseAltered Transcription microRNA Gene Genomic Instability Pri-miRNA Methylation Amplification/Deletion AAAA Histone Modification Translocation Drosha-DGCR8 Transcription Factors Insertional Mutagenesis Pre-miRNA Drosha Processing Exportin DICER-TRBP Dicer Processing Loss of miRNA mature miRNA Ago Binding Site in target SNP or Mutation miRNP Alternative Splicing Loss of 3’-UTR Target Transcript -4- Sample & Assay Technologies
  • 5. Circa 2005: Unique miRNA signatures are found in human cancers miRNAs located in genomic regions amplified in cancers (e.g. miR-12-92 cluster) can function as oncogenes, whereas miRNAs located in portions of chromosomes deleted in cancers (e.g. miR- 15a-miR-16-1 cluster) can function as tumor suppressors. Abnormal expression of miRNAs has been found in both solid and hematopoietic tumors. miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression and response to therapy. The race was on to develop miRNA biomarkers and therapeutics!Array data from Calin and Croce Nature Reviews Cancer 6, 857–866 (2006) -5- Sample & Assay Technologies
  • 6. Circa 2011: A growing picture of miRNA dysregulation in cancerIn the last 5 years, a substantial number of studies and reviews have associated.the presence of various miRNAs with cell proliferation, resistance to apoptosis, and.differentiation in cancer cells..For example;. Deletions of miRNA-regulated genes have been detected in more than 65% of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in 16% to 40% of multiple myelomas, and in 60% of prostate cancers. Other miRNA abnormalities have been reported in a wide variety of Human neoplasms, including other hematologic malignancies such as promyelocytic leukemia; Benign tumors such as leiomyoma and pituitary adenoma Multiple types of carcinomas, including pancreatic, esophageal, thyroid, lung, and breast Neuroblastomas and glioblastomas. -6- Sample & Assay Technologies
  • 7. Therapeutics:miRNA as ‘drug’, miRNA as ‘target of drug’ -7- Sample & Assay Technologies
  • 8. Isolation Quantification Functionalization Purification of circulating miRNA -8- Sample & Assay Technologies
  • 9. miRNA in BloodRBC, WBC, platelets, CTC, ‘other cells’, extracellular? Whole blood contains RBC, WBC, platelets, other cells (e.g. circulating tumor cells) Serum (after clotting) Plasma (no clot) High levels of nucleases present in plasma: Freely circulating RNA should be rapidly degraded Surprisingly, stabile miRNA can be detected in serum and plasma -9- Sample & Assay Technologies
  • 10. Stable miRNA in circulation Exosomes, microvesicles, complexed: An evolving story Ago Ago-2-miRNA miRNA complexes4 HDL mediated Other ‘protective’ Exosomes & miRNA transport5 protein6 micro vesicles1,2,31) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-6592) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e36943) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442- 174524) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc. Natl. Acad. Sci 108: 5003-50085) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:4236) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res. 2010 Nov 1;38(20):7248-59. - 10 - Sample & Assay Technologies
  • 11. miRNA in circulation Exosomes, other vesicles, complexed: An evolving storyA pilot study of circulating miRNAs as potential biomarkers of early stage breast Differential expression of microRNAs in plasma of patients with colorectalA translational study of circulating cell-free microRNA-1 in acute myocardial Direct serum assay for microRNA-21 concentrations in early and advanced breastAltered expression levels of miRNAs in serum as sensitive biomarkers for early Exosomal microRNA: a diagnostic marker for lung cancer.Analysis of circulating microRNA biomarkers in plasma and serum using Exosome isolation for proteomic analyses and RNA profiling.Analysis of Circulating MicroRNA: Preanalytical and Analytical Challenges. Exosomes: proteomic insights and diagnostic potential.Argonaute2 complexes carry a population of circulating microRNAs independent of Extracellular microRNA: A new source of biomarkers.Cell-free miRNAs may indicate diagnosis and docetaxel sensitivity of tumor cells Flow cytometric analysis of circulating microparticles in plasma.Circulating microRNA in body fluid: a new potential biomarker for cancer Functional delivery of viral miRNAs via exosomes.Circulating MicroRNA Is A Biomarker of Pediatric Crohn Disease. Human traumatic brain injury alters plasma microRNA levels.Circulating MicroRNA Signatures of Tumor-Derived Exosomes for Early Diagnosis of Identification of Muscle-Specific MicroRNAs in Serum of Muscular Dystrophy AnimalCirculating microRNA: a novel potential biomarker for early diagnosis of acute Let-7 microRNA family is selectively secreted into the extracellular environmentCirculating microRNA-1 as a potential novel biomarker for acute myocardial Let-7 microRNAs are developmentally regulated in circulating human erythroidCirculating MicroRNA-208b and MicroRNA-499 reflect myocardial damage in Liver-specific microRNA-122 target sequences incorporated in AAV vectorsCirculating microRNAs (miRNA) in Serum of Patients With Prostate Cancer. Measuring circulating miRNAs: the new "PSA" for Breast Cancer?Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Methods for the discovery of low-abundance biomarkers for urinary bladder cancerCirculating microRNAs as biomarkers and potential paracrine mediators of Micromarkers: miRNAs in cancer diagnosis and prognosis.Circulating microRNAs as biomarkers for hepatocellular carcinoma. MicroRNA as biomarkers and regulators in B-cell chronic lymphocytic leukemia.Circulating microRNAs as blood-based markers for patients with primary and MicroRNA dysregulation in gastric cancer: a new player enters the game.Circulating microRNAs as novel minimally invasive biomarkers for breast cancer. MicroRNA expression in human omental and subcutaneous adipose tissue.Circulating microRNAs as potential biomarkers of coronary artery disease: a MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarianCirculating microRNAs as stable blood-based markers for cancer detection. MicroRNA-134 plasma levels before and after treatment for bipolar mania.Circulating microRNAs in breast cancer and healthy subjects. MicroRNAs are transported in plasma and delivered to recipient cells byCirculating microRNAs in patients with coronary artery disease. MicroRNAs as circulating biomarkers for heart failure: questions aboutCirculating microRNAs in plasma of patients with gastric cancers. MicroRNAs as Novel Biomarkers for Breast Cancer.Circulating microRNAs, miR-21, miR-122, and miR-223, in patients with MiR423-5p as a circulating biomarker for heart failure.Circulating microRNAs, possible indicators of progress of rat Pancreatic cancers epigenetically silence SIP1 and hypomethylate and overexpressCirculating microRNAs, potential biomarkers for drug-induced liver injury. Peripheral blood microRNAs distinguish active ulcerative colitis and CrohnsCirculating MicroRNAs: a novel class of biomarkers to diagnose and monitor human Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAsCirculating microRNAs: Association with disease and potential use as biomarkers. Plasma microRNA-122 as a biomarker for viral-, alcohol-, and chemical-relatedCirculating microRNAs: novel biomarkers for cardiovascular diseases? Plasma microRNAs are promising novel biomarkers for early detection of colorectalCirculating microRNAs: novel biomarkers for esophageal cancer. Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury.Circulating microRNAs: possible prediction biomarkers for personalized therapy of Plasma miR-208 as a biomarker of myocardial injury.Circulating microRNAs: promising breast cancer biomarkers - authors response. Recovering circulating extracellular or cell-free RNA from bodily fluids.Circulating microRNAs: promising breast cancer Biomarkers. Relevance of circulating tumor cells, extracellular nucleic acids, and exosomesCirculating miR-210 as a Novel Hypoxia Marker in Pancreatic Cancer. Screening for circulating nucleic acids and caspase activity in the peripheralCirculating miR-221 directly amplified from plasma is a potential diagnostic and Secretory mechanisms and intercellular transfer of microRNAs in living cells.Circulating miR-29a levels in patients with scleroderma spectrum disorder. Secretory microRNAs as a versatile communication tool.Circulating miRNA and cancer diagnosis. Selective release of microRNA species from normal and malignant mammaryCirculating miRNA signatures: promising prognostic tools for cancer. Serum and urinary free microRNA level in patients with systemic lupusCirculating miRNAs are correlated with tumor progression in prostate cancer. Serum microRNA characterization identifies miR-885-5p as a potential marker forCirculating muscle-specific microRNA, miR-206, as a potential diagnostic marker Serum microRNAs are promising novel biomarkers.Circulating nucleic acids as a potential source for cancer biomarkers. Serum microRNAs as non-invasive biomarkers for cancer.Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and Serum miR-146a and miR-223 as potential new biomarkers for sepsis.Detection and characterization of placental microRNAs in maternal plasma. Stable serum miRNA profiles as potential tool for non-invasive lung cancerDetection of circulating fetal nucleic acids: a review of methods and Systemic miRNA-195 differentiates breast cancer from other malignancies and is aDetection of elevated levels of tumour-associated microRNAs in serum of patients The circulating microRNA-221 level in patients with malignant melanoma as a newDetection of microRNA expression in human peripheral blood microvesicles. The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetalDiagnostic and prognostic value of circulating miR-221 for extranodal natural Tissular and soluble miRNAs for diagnostic and therapy improvement in digestiveDiagnostic applications of cell-free and circulating tumor cell-associated miRNAs Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and - 11 - Sample & Assay Technologies
  • 12. miRNeasy MiniPurification of total circulating miRNA Plasma Serum QIAzol Bind Wash Elute Manual or Automated on QIAcube RNeasy Protect Animal Blood PAXgene Blood miRNA Kit - 12 - Sample & Assay Technologies
  • 13. Quantification and profiling of circulating miRNA ncRNA Assays Other small RNAs mRNAs miScript miRNAs System Quantitect Sybr Assays miScript Assays Quantifast Probe Assays RT2 Profiling Arrays RT2 miRNOme Pre-miRNAs Pre-miRNA Assays - 13 - Sample & Assay Technologies
  • 14. miScript system Reverse transcription - principle 2x105 cells 2 cells Linear cDNA synthesis from 10 pg – 1µg RNAmRNA miRNA, other small RNAs - 14 - Sample & Assay Technologies
  • 15. RT2 miRNA PCR ArraysGenome-wide, disease, & pathway-focused analysis miRNome (human, rat, mouse, dog) miFinder abundantly expressed & well-characterized miRNAs General Cancer Cell Development & Differentiation Immunopathology Inflammation Neurological Dev and Disease Brain Cancer Serum and Disease Custom Arrays - 15 - Sample & Assay Technologies
  • 16. Compatible instrumentation: 96- & 384-well formats 96-Well Blocks: 7000, 7300, 7500, 7700, 7900HT, ViiA 7 . iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384 . FAST 96-Well Blocks: 7500, 7900HT, Step One Plus, ViiA 7 . Opticon, Opticon 2, Chromo 4 . FAST 384-Well Block: 7900HT, ViiA 7 . Mastercycler ep realplex 2/2S/4/4S . LightCycler 480 .Mx3000p, Mx3005p, Mx4000p. TP-800 . RotorGene Q . PCR Array Service Core - 16 - Sample & Assay Technologies
  • 17. RT2 miRNA Arrays: Reproducibility of technical replicates The RT2 miRNA PCR Assays are highly reproducible, ensuring plate-to-plate, run-to-run, and sample-to-sample reliability. - 17 - Sample & Assay Technologies
  • 18. Replicates: Technical versus biologicalTechnical Replicates Reproducibility of the PCR Arrays is very high Results demonstrate that what you are seeing is a result of biology, not technique RTC & PPC show technical reproducibility, and can be compared across platesBiological Replicates Needed to verify the results are a result of biology Three biological replicates needed for statistical analysis p values and 95% Confidence Intervals - 18 - Sample & Assay Technologies
  • 19. Genome-wide miRNA profiling with RT2 miRNome Array PC3 cells transduced with p53 vs. empty control vector 40 Ct Adeno-p53 35 30 miR-203 203 551a miR-551a 25 miR-34a 34a miR-940 940 20 15 15 20 25 30 35 40 Ct ControlRT2 Human miRNome PCR Array:Identifies Known and Novel miRNA Targets of the p53 Signaling Pathway - 19 - Sample & Assay Technologies
  • 20. Serum miRNA profilingHuman Serum RT2 miRNA PCR Array (MAH-106). Profile the expression of mature miRNA that researchers have reported as elevated in serum and other bodily fluids in disease Heart and liver injury or disease, atherosclerosis, diabetes, and a number of organ-specific cancers What is on the array? 85 miRNA assays housekeeping gene assays Reverse Transcription Control assays – Monitor RT efficiency PCR Control assays – Monitor PCR efficiency RNA Recovery Control assays – Works with Syn-cel-miR-39 miScript miRNA Mimic (MSY0000010) spiked into samples before nucleic acid preparation to monitor miRNA recovery rates - 20 - Sample & Assay Technologies
  • 21. Human Serum RT2 miRNA PCR Array 1 2 3 4 5 6 7 8 9 10 11 12A let-7a miR-1 miR-100 miR-106a miR-106b miR-10b miR-122 miR-124 miR-125b miR-126 miR-133a miR-133bB miR-134 miR-141 miR-143 miR-146a miR-150 miR-155 miR-17 miR-17* miR-18a miR-192 miR-195 miR-196aC miR-19a miR-19b miR-200a miR-200b miR-200c miR-203 miR-205 miR-208a miR-20a miR-21 miR-210 miR-214D miR-215 miR-221 miR-222 miR-223 miR-224 miR-23a miR-25 miR-27a miR-296-5p miR-29a miR-30d miR-34aE miR-375 miR-423-5p miR-499-5p miR-516a-3p miR-574-3p miR-885-5p miR-9 miR-92a miR-93 let-7c miR-107 miR-10aF miR-128 miR-130b miR-145 miR-148a miR-15a miR-184 miR-193a-5p miR-204 miR-206 miR-211 miR-26b miR-30eG miR-372 miR-373 miR-374a miR-376c miR-7 miR-96 miR-103 miR-15b miR-16 miR-191 miR-22 miR-24H miR-26a cel-miR-39 cel-miR-39 cel-miR-39 SNORD48 SNORD47 SNORD44 RNU6-2 miRTC miRTC PPC PPC Circulating Disease mIRNAs serum miRNAs NC RNA Oncogenic miRNA spike in control RT2 Controls Functional Gene Groupings Injury: Heart Injury: miR-1, miR-133a, miR-192, miR-208a, miR-423-5p, miR-499-5p. Liver Injury: miR-122. Disease: Atherosclerosis: miR-150. Diabetes: miR-124, miR-146a, miR-29a, miR-30d, miR-34a, miR-375, miR-9. Heart Disease: miR-133a, miR-208a. Liver Disease: miR-146a, miR-215, miR-224, miR-574-3p, miR-885-5p, miR-92a. Cancer: Adenocarcinoma: miR-29a, miR-92a. B Cell Lymphoma: miR-21, miR-210. Breast: let-7a, miR-106a, miR-10b, miR-141, miR-155, miR-195, miR-21, miR-34a. Colon: miR-134, miR-146a, miR-17*, miR-221, miR-222, miR-23a, miR-29a, miR-92a. Gastric: let-7a, miR-1, miR-106a, miR-106b, miR-17, miR-17*, miR-20a, miR-21, miR-27a, miR-34a, miR-423-5p. Leukemia: miR-155, miR-21, miR-210. Liver: miR-122, miR-21, miR-223. Lung: miR-134, miR-146a, miR-17*, miR-21, miR-210, miR-221, miR-222, miR-223, miR-23a, miR-25. Ovarian: miR-126, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-21, miR-214, miR-29a, miR-92a, miR-93. Pancreatic: miR-196a, miR-200a, miR-200b, miR-21, miR-210. Prostate: miR-100, miR-125b, miR-141, miR-143, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-296-5p, miR-375, miR-516a-3p Renal: miR-124. Rhabdomyosarcoma: miR-1, miR-133a, miR-133b. - 21 - Sample & Assay Technologies
  • 22. Workflow for circulating miRNA profilingSpike C elegans miRNA control into lysate - 22 - Sample & Assay Technologies
  • 23. Profiling of circulating miRNA Stability of endogenous miRNA in human serum and plasma at 25oC 2 R = 0.945 Serum 35 30 25 Circulating miRNA is not naked, as unprotected miRNA would24 hours 25oC 20 have a half-life of only minutes 15 2 24 hours at ambient temperature R = 0.932 Plasma 35 has little effect on the miRNA profile from serum or plasma 30 25 20 15 15 20 25 30 35 40 0 hours 25oC - 23 - Sample & Assay Technologies
  • 24. Profiling of circulating miRNA Freeze/thaw stability of miRNA in human serum and plasma 2 R = 0.973 Serum 35 30 Circulating miRNA is unaffected by up to 20 freeze-thaw cycles 25 20 Storing multiple archival aliquots20 F/T cycles is suggested for precious 15 samples to avoid mishaps 2 R = 0.984 Plasma 35 Whole blood should NOT be 30 subject to freeze-thaw to avoid cell lysis 25 20 15 15 20 25 30 35 40 1 freeze-thaw cycle - 24 - Sample & Assay Technologies
  • 25. Profiling of circulating miRNA Subtle differences between serum and plasma (same donor) 40.0 R2 = 0.922 35.0 30.0serum 25.0 20.0 15.0 15.0 20.0 25.0 30.0 35.0 40.0 plasma Triplicate isolations from the same plasma or serum sample (not triplicate collections of fluid) Most assays do not vary by more than one Ct Either serum or plasma is suitable for profiling but plasma may be more consistent as the additional variability of clotting is not required Recommendation is to compare serum to serum, plasma to plasma, as normalization will not correct for these outliers. - 25 - Sample & Assay Technologies
  • 26. m iR -1 6 0 5 10 15 20 25 30 35 m iR -9 2a m iR -1 9b m ir- 2 23 m iR -1 26 m iR -2 1 m iR -1 5b le t- 7b m iR -9 3 m iR -1 91 m iR -2 7a le t- 7g le t- 7a m ir- 2 m 4 iR -2 6b m iR -4 25 m iR -1 92 m iR -1 48 m a iR -1 25 m b iR -1 46 a m iR -1 5a m iR -2 9c - 26 - m iR -3 0e le t- 7d m ir- 3 0a miRNeasy Mini and RT2 Array: m iR -1 97 m iR -1 82 m iR -2 9a m iR -1 25 a m iR -2 22 m iR -9 9a m iR -1 52 m iR ~ 20 µl serum per cDNA synthesis (per 384 array) -1 93 a m Profiling of circulating miRNA from plasma or serum iR -1 0b m iR -1 96 a m iR -1 m 83 iR -1 7- 3p m iR -1 42 m iR -1 45 U 6 R N U 1ASample & Assay Technologies
  • 27. Variability in circulating miRNA in normal volunteers34.00 34.00 34.00 y = 0.9098x + 1.8318 y = 0.9092x + 1.9134 y = 0.9707x + 0.928632.00 32.00 32.0030.00 30.00 30.0028.00 28.00 28.0026.00 26.00 26.0024.00 24.00 24.0022.00 22.00 22.0020.00 20.00 20.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 40.00 38.00 36.00 34.00 Title 32.00 30.00 Description 28.00 26.00 Date 24.00 22.00 20.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 - 27 - Sample & Assay Technologies
  • 28. ‘Normal’ expression levels of ‘serum miRNAs’Deviation from mean level of expression, each gene Black = least variable Green = variance above geometric mean Red = variance below geometric mean - 28 - Sample & Assay Technologies
  • 29. ‘Normal’ expression levels of ‘serum miRNAs’ Displayed as relative level of expression, normalized to plate meanGreen = highly expressedRed = low level of expressionBlack = mean expression - 29 - Sample & Assay Technologies
  • 30. Variability in miRNA in RT2 Serum Array Among 10 ‘normal’ volunteers45.0040.0035.00 Less than30.00 2-fold variability25.0020.0015.00 hsa-miR-30e hsa-miR-17 hsa-miR-19b hsa-miR-150 hsa-miR-106a hsa-miR-423-5p hsa-miR-93 hsa-miR-92a hsa-let-7c45.0040.0035.00 >100-fold30.00 Up to 10,000 fold25.00 Variability.20.0015.00 hsa-miR-155 hsa-miR-124 hsa-miR-206 hsa-miR-204 hsa-miR-376c hsa-miR-215 hsa-miR-128 hsa-miR-143 hsa-miR-203 - 30 - Sample & Assay Technologies
  • 31. Profiling of circulating miRNA in cancer RT2 Circulating Array, colon and breast cancer samples 40 40 pooled breast cancer n=3pooled colon cancer n=3 R2 = 0.9067 R2 = 0.9361 38 38 36 36 34 34 32 32 30 30 28 28 26 26 24 24 22 22 20 20 20 22 24 26 28 30 32 34 36 38 40 20 22 24 26 28 30 32 34 36 38 40 pooled normal n=10 pooled normal n=10 37 37 35 35 ‘Unacceptable’ commercial serum Low signal levels Pancreatic Cancer A Prostate Cancer A 33 33 More ‘undetectable’ targets 31 31 High variability between samples 29 29 27 27 25 25 29 31 33 35 37 29 31 33 35 37 Normal Normal - 31 - Sample & Assay Technologies
  • 32. RT2 Dog miRNome serum/plasma profiling33.00 R2 = 0.9731.0029.00 Mirbase v16, 275 genes27.0025.00 Single use 384 RT2 Array23.00 RT and PCR controls21.00 Wet lab validated19.00 miRNeasy serum / plasma protocol17.00 serum vs plasma, same dog 200 ul serum or plasma prep15.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00 33.00 50 ul eluate33.00 5ul or RNA prep for cDNA synthesis R2 = 0.962831.00 one 384 well RT2 PCR Array29.0027.0025.0023.0021.0019.0017.00 serum dog 1 vs dog 215.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00 33.00 - 32 - Sample & Assay Technologies
  • 33. Isolation Quantification Functionalization Circulating miRNA data normalization - 33 - Sample & Assay Technologies
  • 34. Expression profiling of normal human serum samples Data normalization strategies. Two normal human serum samples (Sample A and Sample B) Total RNA was isolated using the miRNeasy Mini Kit QIAGEN Supplementary Protocol for total RNA purification from serum or plasma Option syn-cel-miR-39 spike-in control included 5 µl of each RNA elution was used in an RT2 miRNA First Strand Kit reverse transcription reaction Mature miRNA expression was profiled using the Human Serum RT2 miRNA PCR Array (MAH-106) 40 Non-normalized Ct values are highly comparable Raw Ct: Serum Sample B 35 How should the data be normalized to uncover fine differences between the two samples? 30 25 20 2 R = 0.9079 15 15 20 25 30 35 40 Raw Ct: Serum Sample A - 34 - Sample & Assay Technologies
  • 35. Serum sample and RTPCR data normalizationStep 1: Check reverse transcription control (miRTC) and PCR control (PPC) Ct values. Position Control Ct: Sample A Ct: Sample B H09 miRTC 18.76 18.52 H10 miRTC 18.73 18.64 H11 PPC 19.43 19.61 H12 PPC 19.61 19.76 As determined by the raw Ct values, the reverse transcription and PCR efficiency of both samples are highly comparable Ct values differ by less than 0.25 units - 35 - Sample & Assay Technologies
  • 36. Serum sample data normalization (cont.) Step 2: Observe housekeeping gene Ct values . Position Gene Ct: Sample A Ct: Sample B H05 SNORD48 31.81 32.79 H06 SNORD47 35.00 35.00 H07 SNORD44 35.00 35.00 H08 RNU6-2 35.00 35.00 Housekeeping genes are either not expressed or exhibit borderline detectable expression As is often found with serum samples, standard housekeeping genes cannot be used for data normalization How should you proceed? - 36 - Sample & Assay Technologies
  • 37. Serum sample data normalization (cont.)Four potential data normalization options1. Normalize data of each plate to its RNA Recovery Control Assays (wells H02 to H04) Can only be used if Syn-cel-miR-39 miScript miRNA Mimic (MSY0000010) is spiked into the sample @ nucleic acid preparation2. Normalize data to Ct mean of all expressed targets (targets with Ct < 35) for a given plate3. Normalize data to Ct mean of targets that are commonly expressed in the samples of interest4. Normalize data to ‘0’ Essentially you are relying on the consistency of the quantity and quality of your original RT input - 37 - Sample & Assay Technologies
  • 38. Serum sample data normalization (cont.) Option 1: Normalize to RNA recover control assaysCalculate the average Ct of the cel-miR-39 wells (H02 to H04) Position Control Ct: Sample A Ct: Sample B H02 cel-miR-39 17.84 19.37 H03 cel-miR-39 17.85 19.49 H04 cel-miR-39 17.85 19.39 • Sample A: 17.85 • Sample B: 19.42Using these cel-miR-39 Ct means as normalizers, calculate ∆∆Ctvalues, fold-change, and fold up/down regulation 100 80 Fold-Regulation (B to A) 60 40 20 0 -20 -40 - 38 - Sample & Assay Technologies
  • 39. Serum sample data normalization (cont.) Option 2: Normalize to Ct mean of expressed targets for a given plateDetermined the number of expressed targets in each plate (Ct < 35) Sample A: 66 Sample B: 59Calculate the Ct Mean of the expressed targets Sample A: 28.96 Sample B: 29.70Using these Ct means as normalizers, calculate ∆∆Ct values, fold-change,and fold up/down regulation NOTE: same strongly up-regulated and down-regulated miRNAs are identified 60 40 Fold-Regulation (B to A) 20 0 -20 -40 -60 - 39 - Sample & Assay Technologies
  • 40. Serum sample data normalization (cont.) Option 3: Normalize to Ct mean of commonly expressed targetsDetermined the number of commonly expressed targets for the plates beinganalyzed (Ct < 35 in all samples) Commonly expressed in Sample A and Sample B: 48Calculate the associated Ct Mean Sample A: 27.52 Sample B: 28.86Using these Ct means as normalizers, calculate ∆∆Ct values, fold-change,and fold up/down regulation NOTE: same strongly up-regulated and down-regulated miRNAs are identified 80 60 Fold-Regulation (B to A) 40 20 0 -20 -40 - 40 - Sample & Assay Technologies
  • 41. Serum sample data normalization (cont.) Option 4: Normalize to ‘0’Normalizing to ‘0’ relies on the consistency in the quantity and quality ofyour original RT input For serum samples, this may not be the best option, as the RNA is not routinely quantified prior to addition to a reverse transcription reactionNormalizing the data to ‘0’, calculate ∆∆Ct values, fold-change, and foldup/down regulation 40 20 Fold-Regulation (B to A) 0 -20 -40 -60 -80 -100NOTE: These results are not completely comparable to the results achieved with theother three normalization methods. The same strongly up-regulated and down-regulated miRNAs are identified; however, additionally up- and down-regulatedgenes are potentially (incorrectly) identified. This suggests that there is the need forsome method of normalization, other than just normalizing to ‘0’. - 41 - Sample & Assay Technologies
  • 42. www.qiagen.com/geneglobe - 42 - Sample & Assay Technologies
  • 43. QUESTIONS?Webinar 1 : Biogenesis, function & analysis of miRNA & its role in human diseaseWebinar 2 : miRNAs and Cancer: The role of miR-211 in MelanomaWebinar 3 : miRNA Profiling in serum & plasma On the road to biomarker development Thank you! Eric Lader eric.lader@qiagen.com - 43 - Sample & Assay Technologies