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 -
RNA interference: A natural phenomenon Discovery tool, potential therapeutic May 26, 2003-2 -
Canonical pathway of microrna biogenesis NUCLEUS microrna Gene POL II DNA Pri-miRNA Transcribed by RNA Polymerase II as a long primary transcript (pri-mirnas), which may contain more than one mirna. CYTOPLASM Drosha-DGCR8 Exportin Pre-miRNA In the nucleus, Pri-miRNAs are processed to hairpin-like pre-mirnas by RNAse IIIlike enzyme Drosha Exportin Pre-miRNAs are then exported to the Cytosol by Exportin 5 DICER-TRBP mature mirna In the cytosol RNAse III-like Dicer, processes these precursors to mature mirnas RISC Ago RISC Assembly These mirnas are incorporated in RISC High 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 degradation Krol, J et.al., (2010) Nature Rev Genetics, 11, 597; Winter, J. et.al., (2009) Nature Cell Biology, 11, 228-3 -
Potential events leading to disruption of normal mirna:target interaction in disease Altered Transcription Methylation Histone Modification Transcription Factors Drosha Processing microrna Gene Pri-miRNA Drosha-DGCR8 Exportin AAAA Pre-miRNA Genomic Instability Amplification/Deletion Translocation Insertional Mutagenesis Dicer Processing DICER-TRBP mature mirna mirnp Target Transcript Ago Loss of mirna Binding Site in target SNP or Mutation Alternative Splicing Loss of 3 -UTR - 4 -
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, 857 866-5 - (2006)
Circa 2011: A growing picture of mirna dysregulation in cancer.in 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 -
Therapeutics: mirna as drug, mirna as target of drug - 7 -
Isolation Quantification Functionalization Purification of circulating mirna - 8 -
mirna in Blood RBC, 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 -
Stable mirna in circulation Exosomes, microvesicles, complexed: An evolving story Ago mirna Exosomes & micro vesicles 1,2,3 Ago-2-miRNA complexes 4 HDL mediated mirna transport 5 Other protective protein 6 1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9:654-659 2) Hunter MP et. al., (2008) Detection of microrna Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694 3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of micrornas in living cells, J Biol Chem 285: 17442-17452 4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating micrornas independent of vesicles in human plasma, Proc. Natl. Acad. Sci 108: 5003-5008 5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423 6) 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 -
mirna in circulation Exosomes, other vesicles, complexed: An evolving story A pilot study of circulating mirnas as potential biomarkers of early stage breast Differential expression of micrornas in plasma of patients with colorectal A translational study of circulating cell-free microrna-1 in acute myocardial Direct serum assay for microrna-21 concentrations in early and advanced breast Altered 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 Animal Circulating microrna: a novel potential biomarker for early diagnosis of acute Let-7 microrna family is selectively secreted into the extracellular environment Circulating microrna-1 as a potential novel biomarker for acute myocardial Let-7 micrornas are developmentally regulated in circulating human erythroid Circulating MicroRNA-208b and MicroRNA-499 reflect myocardial damage in Liver-specific microrna-122 target sequences incorporated in AAV vectors Circulating 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 cancer Circulating 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 ovarian Circulating 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 by Circulating micrornas in patients with coronary artery disease. MicroRNAs as circulating biomarkers for heart failure: questions about Circulating 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 overexpress Circulating micrornas, potential biomarkers for drug-induced liver injury. Peripheral blood micrornas distinguish active ulcerative colitis and Crohn's Circulating MicroRNAs: a novel class of biomarkers to diagnose and monitor human Plasma microrna profiling reveals loss of endothelial mir-126 and other micrornas Circulating micrornas: Association with disease and potential use as biomarkers. Plasma microrna-122 as a biomarker for viral-, alcohol-, and chemical-related Circulating micrornas: novel biomarkers for cardiovascular diseases? Plasma micrornas are promising novel biomarkers for early detection of colorectal Circulating 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 exosomes Circulating mir-210 as a Novel Hypoxia Marker in Pancreatic Cancer. Screening for circulating nucleic acids and caspase activity in the peripheral Circulating 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 mammary Circulating mirna signatures: promising prognostic tools for cancer. Serum and urinary free microrna level in patients with systemic lupus Circulating mirnas are correlated with tumor progression in prostate cancer. Serum microrna characterization identifies mir-885-5p as a potential marker for Circulating 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 cancer Detection of circulating fetal nucleic acids: a review of methods and Systemic mirna-195 differentiates breast cancer from other malignancies and is a Detection of elevated levels of tumour-associated micrornas in serum of patients The circulating microrna-221 level in patients with malignant melanoma as a new Detection of microrna expression in human peripheral blood microvesicles. The levels of hypoxia-regulated micrornas in plasma of pregnant women with fetal Diagnostic and prognostic value of circulating mir-221 for extranodal natural Tissular and soluble mirnas for diagnostic and therapy improvement in digestive Diagnostic applications of cell-free and circulating tumor cell-associated mirnas Vesicle-related micrornas in plasma of nonsmall cell lung cancer patients and - 11 -
mirneasy Mini Purification of total circulating mirna Plasma Serum QIAzol Bind Wash Elute Manual or Automated on QIAcube RNeasy Protect Animal Blood PAXgene Blood mirna Kit - 12 -
Quantification and profiling of circulating mirna ncrna Assays Other small RNAs mrnas Quantitect Sybr Assays Quantifast Probe Assays miscript System mirnas miscript Assays RT 2 Profiling Arrays RT 2 mirnome Pre-miRNAs Pre-miRNA Assays - 13 -
miscript system Reverse transcription - principle 2x10 5 cells 2 cells Linear cdna synthesis from 10 pg 1µg RNA mrna mirna, other small RNAs - 14 -
RT 2 mirna PCR Arrays Genome-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 -
Compatible instrumentation: 96- & 384-well formats.96-well Blocks: 7000, 7300, 7500, 7700, 7900HT, ViiA 7.FAST 96-Well Blocks: 7500, 7900HT, Step One Plus, ViiA 7.FAST 384-Well Block: 7900HT, ViiA 7.iCycler, MyiQ, MyiQ2, iq5, CFX96, CFX384.Opticon, Opticon 2, Chromo 4.Mastercycler ep realplex 2/2S/4/4S.LightCycler 480.Mx3000p, Mx3005p, Mx4000p.TP-800.RotorGene Q PCR Array Service Core - 16 -
RT 2 mirna Arrays: Reproducibility of technical replicates The RT 2 mirna PCR Assays are highly reproducible, ensuring plate-to-plate, run-to-run, and sample-to-sample reliability. - 17 -
Replicates: Technical versus biological Technical 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 plates Biological 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 -
Genome-wide mirna profiling with RT 2 mirnome Array PC3 cells transduced with p53 vs. empty control vector 40 35 Ct Adeno-p53 30 25 20 mir-34a mir-940 34a 940 203 mir-203 mir-551a 15 15 20 25 30 35 40 Ct Control RT 2 Human mirnome PCR Array: Identifies Known and Novel mirna Targets of the p53 Signaling Pathway - 19 -
Serum mirna profiling.human Serum RT 2 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 -
Human Serum RT 2 mirna PCR Array 1 2 3 4 5 6 7 8 9 10 11 12 A let-7a mir-1 mir-100 mir-106a mir-106b mir-10b mir-122 mir-124 mir-125b mir-126 mir-133a mir-133b B mir-134 mir-141 mir-143 mir-146a mir-150 mir-155 mir-17 mir-17* mir-18a mir-192 mir-195 mir-196a C mir-19a mir-19b mir-200a mir-200b mir-200c mir-203 mir-205 mir-208a mir-20a mir-21 mir-210 mir-214 D mir-215 mir-221 mir-222 mir-223 mir-224 mir-23a mir-25 mir-27a mir-296-5p mir-29a mir-30d mir-34a E 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-10a F mir-128 mir-130b mir-145 mir-148a mir-15a mir-184 mir-193a-5p mir-204 mir-206 mir-211 mir-26b mir-30e G mir-372 mir-373 mir-374a mir-376c mir-7 mir-96 mir-103 mir-15b mir-16 mir-191 mir-22 mir-24 H 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-3 Renal: mir-124. Rhabdomyosarcoma: mir-1, mir-133a, mir-133b. - 21 -
Workflow for circulating mirna profiling Spike C elegans mirna control into lysate - 22 -
Profiling of circulating mirna Stability of endogenous mirna in human serum and plasma at 25 o C 35 R 2 = 0.945 Serum 30 24 hours 25 o C 25 20 15 35 R 2 = 0.932 Plasma Circulating mirna is not naked, as unprotected mirna would have a half-life of only minutes 24 hours at ambient temperature has little effect on the mirna profile from serum or plasma 30 25 20 15 15 20 25 30 35 40 0 hours 25 o C - 23 -
Profiling of circulating mirna Freeze/thaw stability of mirna in human serum and plasma 35 R 2 = 0.973 Serum 30 25 Circulating mirna is unaffected by up to 20 freeze-thaw cycles 20 F/T cycles 20 15 35 30 R 2 = 0.984 Plasma Storing multiple archival aliquots is suggested for precious samples to avoid mishaps Whole blood should NOT be subject to freeze-thaw to avoid cell lysis 25 20 15 15 20 25 30 35 40 1 freeze-thaw cycle - 24 -
Profiling of circulating mirna Subtle differences between serum and plasma (same donor) 40.0 35.0 R 2 = 0.922 serum 30.0 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 -
35 30 25 20 15 10 5 0 mirneasy Mini and RT 2 Array: Profiling of circulating mirna from plasma or serum ~ 20 µl serum per cdna synthesis (per 384 array) mir-21 mir-15b let-7b mir-93 mir-191 mir-27a let-7g let-7a mir-24 mir-26b mir-425 mir-192 mir-148a mir-125b mir-146a mir-15a mir-29c mir-30e let-7d mir-30a mir-197 mir-182 mir-29a mir-125a mir-222 mir-99a mir-152 mir-193a mir-10b mir-196a mir-183 mir-17-3p mir-142 mir-145 U6 RNU1A mir-126-26 - mir-223 mir-19b mir-92a mir-16
Variability in circulating mirna in normal volunteers 34.00 32.00 y = 0.9098x + 1.8318 34.00 32.00 y = 0.9092x + 1.9134 34.00 32.00 y = 0.9707x + 0.9286 30.00 30.00 30.00 28.00 28.00 28.00 26.00 26.00 26.00 24.00 24.00 24.00 22.00 22.00 22.00 20.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 20.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 20.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 40.00 38.00 36.00 34.00 32.00 30.00 28.00 26.00 24.00 Title Description Date 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 -
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 -
Normal expression levels of serum mirnas Displayed as relative level of expression, normalized to plate mean Green = highly expressed Red = low level of expression Black = mean expression - 29 -
Variability in mirna in RT 2 Serum Array Among 10 normal volunteers 45.00 40.00 35.00 30.00 Less than 2-fold variability 25.00 20.00 15.00 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-7c 45.00 40.00 35.00 >100-fold 30.00 25.00 Up to 10,000 fold Variability. 20.00 15.00 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 -
Profiling of circulating mirna in cancer RT 2 Circulating Array, colon and breast cancer samples pooled colon cancer n=3 40 R 2 = 0.9067 38 36 34 32 30 28 26 24 22 20 20 22 24 26 28 30 32 34 36 38 40 pooled breast cancer n=3 40 R 2 = 0.9361 38 36 34 32 30 28 26 24 22 20 20 22 24 26 28 30 32 34 36 38 40 pooled normal n=10 pooled normal n=10 Prostate Cancer A 37 35 33 31 29 27 Pancreatic Cancer A 37 35 33 31 29 27 Unacceptable commercial serum Low signal levels More undetectable targets High variability between samples 25 29 31 33 35 37 Normal 25 29 31 33 35 37 Normal - 31 -
RT 2 Dog mirnome serum/plasma profiling 33.00 31.00 R 2 = 0.97 29.00 27.00 25.00 23.00 21.00 19.00 17.00 serum vs plasma, same dog 15.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00 33.00 33.00 R 2 = 0.9628 31.00 Mirbase v16, 275 genes Single use 384 RT 2 Array RT and PCR controls Wet lab validated mirneasy serum / plasma protocol 200 ul serum or plasma prep 50 ul eluate 5ul or RNA prep for cdna synthesis one 384 well RT 2 PCR Array 29.00 27.00 25.00 23.00 21.00 19.00 17.00 serum dog 1 vs dog 2 15.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00 33.00-32 -
Isolation Quantification Functionalization Circulating mirna data normalization - 33 -
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 RT 2 mirna First Strand Kit reverse transcription reaction Mature mirna expression was profiled using the Human Serum RT 2 mirna PCR Array (MAH-106) 40 Non-normalized Ct values are highly comparable How should the data be normalized to uncover fine differences between the two samples? Raw Ct: Serum Sample B 35 30 25 20 R 2 = 0.9079 15 15 20 25 30 35 40 Raw Ct: Serum Sample A - 34 -
Serum sample and RTPCR data normalization.step 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 -
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 -
Serum sample data normalization (cont.) Four potential data normalization options 1. 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 preparation 2. Normalize data to Ct mean of all expressed targets (targets with Ct < 35) for a given plate 3. Normalize data to Ct mean of targets that are commonly expressed in the samples of interest 4. Normalize data to 0 Essentially you are relying on the consistency of the quantity and quality of your original RT input - 37 -
Serum sample data normalization (cont.) Option 1: Normalize to RNA recover control assays Calculate 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: 19.42 Using these cel-mir-39 Ct means as normalizers, calculate Ct values, fold-change, and fold up/down regulation 100 Fold-Regulation (B to A) 80 60 40 20 0-20 -40-38 -
Serum sample data normalization (cont.) Option 2: Normalize to Ct mean of expressed targets for a given plate Determined the number of expressed targets in each plate (Ct < 35) Sample A: 66 Sample B: 59 Calculate the Ct Mean of the expressed targets Sample A: 28.96 Sample B: 29.70 Using 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 Fold-Regulation (B to A) 40 20 0-20 -40-60 - 39 -
Serum sample data normalization (cont.) Option 3: Normalize to Ct mean of commonly expressed targets Determined the number of commonly expressed targets for the plates being analyzed (Ct < 35 in all samples) Commonly expressed in Sample A and Sample B: 48 Calculate the associated Ct Mean Sample A: 27.52 Sample B: 28.86 Using 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 Fold-Regulation (B to A) 60 40 20 0-20 -40-40 -
Serum sample data normalization (cont.) Option 4: Normalize to 0 Normalizing to 0 relies on the consistency in the quantity and quality of your 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 reaction Normalizing the data to 0, calculate Ct values, fold-change, and fold up/down regulation 40 Fold-Regulation (B to A) 20 0-20 -40-60 -80-100 NOTE: These results are not completely comparable to the results achieved with the other three normalization methods. The same strongly up-regulated and downregulated mirnas are identified; however, additionally up- and down-regulated genes are potentially (incorrectly) identified. This suggests that there is the need for some method of normalization, other than just normalizing to 0. - 41 -
www.qiagen.com/geneglobe - 42 -
QUESTIONS? Webinar 1 : Biogenesis, function & analysis of mirna & its role in human disease Webinar 2 : mirnas and Cancer: The role of mir-211 in Melanoma Webinar 3 : mirna Profiling in serum & plasma On the road to biomarker development Thank you! Eric Lader eric.lader@qiagen.com - 43 -