microRNA Sample and Assay Technoligies ( /2012)
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- れんま たけはな
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1 microrna Sample and Assay Technologies mirna Sample & Assay Technologies
2 mirna mirnome mirna micrornas mirnas noncoding RNA 22 1 mirna mirna mirna mirna mirna QIAGEN mirna QIAGEN GeneGlobe mirna QIAGEN RNA Pol II transcription mirna mirneasy Kits PAXgene Kits RNA Drosha Pri-miRNA mirna 5 DGCR8 AAA AAA 3 mirna miscript mirna PCR Arrays mirnome mirna Exportin-5 Pre-miRNA mirna miscript PCR System mature mirnas miscript Primer Assays precursor mirnas miscript Precursor Assays mrna Exportin-5 QuantiTect Primer Assays Dicer TRBP Pre-miRNA mirna miscript mirna Mimics Inhibitors Protectors Mature mirna mirna RISC assembly High homology mrna binding Partial homology Sequence-specific mrna cleavage Translational repression and/or mrna degradation 1. mirna primary mirnas pri-mirnas Drosha DGCR8 precursor mirna stem loops pre-mirnas pre-mirna Exportin- 5-Ran-GTP Dicer TRBP 22 nt mature mirna mature mirna RISC RNA-induced silencing complexes Ago2 RISC mirna mrna 3' mrna mrna mrna mrna mrna mirna 2 mirna Brochure 05/12
3 mirna miscript PCR System mirna miscript PCR System SYBR Green PCR mirna miscript mirna PCR Arrays mirnome mature mirna mirna mature mirnas precursor mirnas small noncoding RNAs ncrna GeneGlobe miscript PCR System mirnome PCR mature mirna cdna mature mirna precursor mirna ncrna mrna miscript mirna PCR Array mirna miscript PCR System RNA cdna mirna PCR mirna miscript PCR System miscript II RT Kit miscript HiSpec Buffer cdna miscript HiFlex Buffer cdna PCR mature mirna PCR mature mirna precursor mirna ncrna mrna 2. miscript II RT Kit miscript mirna PCR Array miscript Primer Assay mature mirna miscript HiSpec Buffer cdna cdna mature mirna precursor mirna noncoding RNA ncrna mrna Primer Assay cdna miscript HiFlex Buffer miscript II RT Kit miscript II RT Kit 1 1 PCR mature mirna RNA 2 miscript HiSpec Buffer mature mirnas cdna mature mirna miscript HiFlex Buffer RNA mature mirna precursor mirna noncoding RNA mrna cdna mirna mirna mrna miscript II RT Kit mirna Brochure 05/12 Sample & Assay Technologies 3
4 miscript SYBR Green PCR Kit miscript SYBR Green PCR Kit QuantiTect SYBR Green PCR Master Mix miscript Primer Assays mature mirna forward mature mirna miscript Universal Primer reverse PCR PCR mirna miscript SYBR Green PCR Kit miscript Primer Assays miscript Precursor Assays miscript Primer Assay mature mirna forward miscript Universal Primer reverse miscript Primer Assays mature mirna miscript Precursor Assay precursor mirna forward reverse precursor mirna mirbase GeneGlobe miscript Assays miscriptprecursor miscript mirna PCR Arrays mature mirna forward miscript Primer Assays miscript mirna PCR Arrays mirnome miscript SYBR Green PCR Kit miscript mirna PCR Arrays mature mirna miscript mirna PCR Array mature mirna miscript mirna PCR Arrays miscript mirna PCR Arrays miscript PCR Controls miscript PCR Controls miscript PCR System mirna PCR miscript PCR Controls 5 snornas 1 snrna miscript mirna PCR Array Data Analysis Tool miscript mirna PCR Arrays miscript mirna PCR Array C T PCR C T mirna Brochure 05/12
5 mirna miscript mirna PCR Arrays mirna miscript mirna PCR Arrays mature mirna forward miscript Primer Assays mir- Nome 1 PCR well Rotor-Disc 2 3 miscript mirna PCR Arrays PCR miscript mirna PCR Array PCR mature mirna 1. miscript mirna PCR Arrays Complete mirnome mifinder Brain Cancer Breast Cancer A B C D E F Cancer PathwayFinder G Cell Differentiation & Development H Ce Ce SN1 SN2 SN3 SN4 SN5 SN6 mirtc mirtc PPC PPC Immunopathology Inflammatory Response & Autoimmunity Neurological Development & Disease Ovarian Cancer Serum & Plasma Custom Array 2. miscript mirna PCR Arrays PCR C. elegans mir-39 miscript Primer Assay snorna/snrna miscript PCR Controls Reverse Positive transcription PCR control control 3. miscript mirna PCR Array miscript mirna PCR Arrays 84 mirna miscript Primer Assays PCR 6 miscript PCR Control 10 C. elegans mir-39 Primer Assay cel-mir-39 spike-in control Syn-cel-miR-39 miscript mirna Mimic Rotor- Disc 384 Manufacturer Cycler Format QIAGEN Rotor-Gene Q Rotor-Gene 6000 R Applied Biosystems ABI Standard 7900HT Standard 96-well block 7700 ViiA 7 96-well block A ABI 7500 Fast 7900HT Fast 96-well block ViiA 7 Fast 96-well block StepOnePlus C ABI 7900HT ViiA well block E Bio-Rad icycler iq 5 MyiQ MyiQ2 Chromo4 A CFX96 DNA Engine Opticon DNA Engine Opticon 2 D CFX384 E Agilent Stratagene Mx00P Mx005P A Mx4000 D Roche LightCycler well block F LightCycler well block G Eppendorf Mastercycler ep realplex 2 2S 4 4S A TaKaRa TP-800 A mirna Brochure 05/12 Sample & Assay Technologies 5
6 mirna cdna 5' 5' RNA sample 1 RNA sample 2 mirna 3' Poly(A) tail 3' A A A A A(A) n 5' 3' A A A A A(A) Single-tube reaction n T T T T T T Polyadenylation Universal RT primer miscript Primer Assay miscript Universal Primer PCR Reverse transcription First strand cdna T T T T T T 5' 3' 2. cdna miscript Universal Primer QuantiTect SYBR Green PCR Mastermix miscript mirna PCR Array miscript Primer Assay T T T T T T 5' 3' cdna miscript Universal Primer 3. PCR miscript mirna PCR Arrays mirna 4 miscript HiSpec Buffer miscript II RT Kit cdna cdna miscript Universal Primer QuantiTect SYBR Green PCR Master Mix RNase miscript mirna PCR Array PCR miscript mirna PCR Array Data Analysis Tool mirna mirna miscript mirna PCR Arrays miscript mirna PCR Arrays miscript mirna PCR Arrays mirna mirnas mirnas mirna mirna 84 Serum & Plasma miscript mirna PCR Array 5 4. Sample Sample 1 4. miscript mirna PCR Array C T value Serum isolation 1 Serum isolation 2 Serum isolation mirna 2 -ΔCT Breast cancer serum 1.E+03 1.E+02 1.E+01 1.E+00 1.E-01 1.E-02 1.E-03 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E ΔCT Normal serum 5. mirna n=10n=3 RNA miscript II RT Kit miscript HiSpec Buffer cdna PCR mature mirna miscript SYBR Green PCR Kit Human Serum & Plasma miscript mirna A 3 C T B 2 -ΔCT mature mirna ±3 6 mirna Brochure 05/12
7 FFPE mirna FFPE mirna QIAGEN FFPE RNA mirneasy FFPE Kit 38 mirna miscript mirna PCR Arrays FFPE mirna 6 26 technical replicate 2 R mirna 2 = mirnome miscript 14 mirna PCR Arrays FFPE isolation 2 15 FFPE isolation µg RNA Mean C mirna mirnome T technical replicate 1 mirna 7 C T value y = x FFPE isolation 1 technical replicate 2 C T value 2 -ΔCT Tumor lung FFPE tissue Mean technical C T biological replicate replicate E mirna y = x R 2 = aza-2'-dC treated HCT 116 cells E-01 1.E-02 1.E E-03 1.E E-04 1.E E-01 1.E-02 1.E-03 1.E-04 1.E-05 1.E-05 1.E-04 1.E-03 1.E-02 1.E ΔCT Normal lung FFPE tissue 1.E-03 1.E-02 1.E-01 1.E+00 Untreated HTC 116 cells FFPE isolation 1 FFPE isolation 2 FFPE isolation 3 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+01 1.E+02 Mean technical C T biological replicate replicate 2 2 y = x ΔCT Normal lung FFPE R 2 tissue y = x FFPE mirna R 2 = E FFPE 5 µm 15 mirneasy FFPE Kit RNA 1.E+03 miscript II RT Kit 15 miscript HiSpec Buffer cdna PCR mature mirna E+02 Human Mean mifinder C miscript mirna PCR Array miscript SYBR Green PCR Kit T technical replicate 1 15 Mean C T biological replicate 1 A 3 FFPE C T B 2 -ΔCT 1.E+01 Mean C 2 mature mirna T technical replicate 1 1.E+00 ±3 biological replicate Log 2 (fold regulation) y = x R 2 = mirna 5-aza-2'-deoxycytidine 5-aza-2'-dC demethylation HCT 116 mirneasy Mini Kit RNA 15 Mean C T biological DNA replicate 1 15 DNA methyltransferase DNA Mean methyltransferase C T technical replicate miscript 1 Mean II RT C T biological Kit miscript replicate HiSpec 1 Buffer cdna PCR mature mirna Human mirnome miscript mirna PCR Array miscript SYBR Green PCR Kit A 2 -ΔCT 2 mature mirna B mirna ±4 5-aza-2'-dC HCT mirnas p mirna 89 mirna ΔCT Tumor lung FFPE tissue p value y = x R y = x R 2 = E+04 1.E+03 1.E+02 1.E+01 1.E+00 1.E-01 1.E-02 1.E E-04 1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 ical replicate 2 mirna Brochure 05/12 Sample & Assay Technologies 7
8 PCR miscript mirna PCR Array Excel C T 8 8. ΔC T (Relative to RNU6-2) let-7a-1 Pre-miRNA Mature mirna let-7a-2 let-7a-3 mir-24-2 mir-27a mir mir-221 mir-18a mir mature mirna precursor mirna mirneasy Mini Kit HeLa S3 RNA miscript II RT Kit miscript HiFlex Buffer cdna miscript Primer Assays mature mirna miscript Precursor Assays precursor mirnas PCR RNU6-2 miscript PCR Control mature mirna precursor mirna mirna precursor mirna mature mirna miscript mirna PCR Array Data Analysis Tools ΔΔC T fold-change mirna precursor mirna mature mirna cdna mirna miscript II RT Kit miscript HiFlex Buffer cdna miscript Primer Assays mature mirna miscript Precursor Assays precursor mirnas PCR 9 miscript Primer Assays miscript Precursor Assays GeneGlobe mature mirna precursor mirna mirna mature mirnas precursor mirnas 9 miscript PCR Starter Kit miscript PCR System mirna mirna miscript PCR Starter Kit PCR mir-15a miscript Primer Assay RNU6-2 miscript PCR Control mirna 8 mirna Brochure 05/12
9 mirna mirna 100 mirna miscript PCR System mirna mirna miscript HiSpec Buffer miscript HiFlex Buffer cdna 10 mirna pg RNA miscript PCR System 1 mirna 3 miscript PCR System Log copy number mirna (miscript HiFlex Buffer) mirna miscript II RT Kit miscript HiFlex Buffer mir-21 cdna cdna miscript PCR System PCR mirna C T miscript HiSpec Buffer 3. miscript Primer Assays cdna used % Activity relative to perfect match primer assay as 100% miscript Primer Assay used Let-7b Let-7c mir-98 Let-7d Let-7e Let-7a Let-7f Let-7g Let-7i Let-7b Let-7c mir Let-7d Let-7e Let-7a Let-7f technical replicate 2 y = x R 2 = Let-7g Let-7i technical replicate 1 Let-7 1 Let-7 Let-7a Let-7i mir-98 mirna miscript II RT Kit miscript HiFlex Buffer cdna miscript HiSpec Buffer cdna miscript Primer Assay PCR miscript Primer Assay miscript Primer Assays C T relative detection = 2 -ΔCT x 100 miscript PCR System 11. RNA mirneasy Mini Kit A HeLa S3 2 B 2 HeLa S3 miscript II RT Kit miscript HiSpec Buffer cdna mifinder miscript mirna PCR Array mirna C T miscript PCR System biological replicate 2 y = x R 2 = biological replicate 1 mirna Brochure 05/12 Sample & Assay Technologies 9
10 QIAGEN mirna RNA Isolation Service miscript mirna PCR Array Service QIAGEN mirna QIAGEN mirna Services QIAGEN mirneasy PAXgene Kits RNA miscript II RT Kit miscript mirna PCR Arrays mirnome miscript mirna PCR Array Data Analysis Tool* * 12 6 Tel mirna PCR mirna RNA mirna miscript PCR Controls miscript PCR System PCR mirna miscript PCR Controls Brain Kidney Liver Lung Skeletal muscle Testis Thymus 100 miscript PCR Controls 5 snornas 1 snrna RNA 12 miscript PCR Controls 100 miscript HiSpec Buffer miscript HiFlex Buffer cdna 5 Brain Kidney Liver Lung Skeletal muscle Testis Thymus miscript PCR Controls miscript PCR Controls miscript mirna PCR Arrays 3 GeneGlobe SNORD61 SNORD68 SNORD72 SNORD95 SNORD96A RNU RNA RNA 0 ng miscript II RT Kit A miscript HiSpec Buffer B miscript HiFlex Buffer cdna miscript PCR System miscript PCR Control 1 ng cdna PCR C T RNA 10 mirna Brochure 05/12
11 mirna mirna mirna mirna mirna miscript mirna Mimics miscript mirna Inhibitors miscript Protectors mirna miscript mirna Mimics mirna miscript mirna Inhibitors mirna miscript Protectors mirna 1 A miscript mirna Mimic mrna A mirna or mimic B miscript mirna Inhibitor mrna A mirna inhibitor C miscript Protector mirna mrna A target protector mrna B mrna B mrna B mrna C mrna C mrna C protein expressed protein not expressed 13. miscript mirna Mimics miscript mirna Inhibitors miscript Protectors A mirnas miscript mirna Mimics mrna B miscript mirna Inhibitors mirna mirna C miscript Protector mirna miscript mirna Mimics mirna miscript mirna Mimics mirna RNA 13 miscript mirna Mimics mirna mirna miscript mirna Mimic HeLa S3 mir-1 mir-16 HiPerFect Transfection Reagent HeLa S3 6 x ' UTR mir-1 3' UTR mir-1 mir-1 Mimic 3' UTR mir mir-1 Mimic mir-16 mirna Brochure 05/12 Sample & Assay Technologies 11
12 miscript mirna Inhibitors mirna miscript mirna Inhibitors RNA mirna miscript mirna Inhibitors mirna miscript mirna Inhibitors 15. mirna miscript mirna Inhibitor HeLa S3 mir-1 mir-16 3' UTR mir-16 mir-16 Inhibitor HeLa S3 6 x 10 4 HiPerFect Transfection Reagent mir-16 Inhibitor mir-1 Inhibitor mirna WT mir-16 Inhibitor mir-16 mir-16 Inhibitor miscript Protector mirna miscript Protectors RNA mirna 1 mirna miscript Protector miscript Protector mrna mirna mirna mirna miscript Protector mirna Untransfected Mimic Mimic + target protector 16. mirna mir-16 MCF-7 mir-16 miscript Protector mirna Neg. control Negative Control miscript Protector Unregulated mir-16 Attractene Transfection Reagent 17. miscript Protector mir-9 MCF-7 mir-9 miscript mirna Mimic 10 nm mir-9 miscript mirna Mimic 10 nm miscript Protector mir-9 1 µm 72 HiPerFect Transfection Reagent mir-9 miscript mirna Mimic mir-9 miscript Protector mir-9 miscript mirna Mimic miscript Protector mir mirna Brochure 05/12
13 mirna RNA small RNA mirna RNA mirna mirna mirneasy PAXgene mirna Kits RNA 4 mirna mirna mirna QIAcube QIAsymphony mirna mirneasy PAXgene Kits RNA RNA mirneasy Precipitation 4. mirna C T value mirneasy Mini Kit 14 1x10 6 1x10 5 1x10 4 1x10 3 1x10 2 mirneasy 96 Kit Cell number mirneasy Micro Kit mirneasy Serum/Plasma Kit mirneasy FFPE Kit PAXgene Blood mirna Kit* PAXgene Tissue mirna Kit FFPE PAXgene Blood RNA Tube PAXgene Tissue Container * QIAsymphony mirna QIAsymphony PAXgene Blood RNA Kit 18. mirneasy Kit Jurkat mirneasy Mini Kit RNA mirna RNA RNA mirna mir-16 RT-PCR mirneasy Kit RNA C T mirneasy Kit mirna mirneasy Kit 1 x 10 2 mirna 1 x PCR mirna RNA mirna mirneasy PAXgene mirna Kits 18 RNA RNA FFPE RNA RNA mirneasy Kits mirna <~0 nt RNA >~0 nt mirneasy PAXgene mirna Kits RNA RT-PCR RNAs miscript mirna PCR Arrays miscript Primer Assays OD mirneasy Precipitation RNA mirna RNA Jurkat 1 x 10 6 mirneasy Mini Kit RNA mirneasy Kit RNA OD 260 nm 260 nm OD 2 nm OD RNA mirna Brochure 05/12 Sample & Assay Technologies 13
14 C T value C T value Liver mir-16 PGK1 Spleen Thymus Kidney Brain mir-16 mir-29a PGK1 24 h Heart Intestine Lung Muscle Pancreas. mirna RNA RNAlater mg mirneasy 96 Kit mirna RNA mir-16 PGK1 RT-PCR RNA PGK1 mrna RNA mirna mir h 21. FFPE mirna RT-PCR mirneasy FFPE Kit mirna RNA mirna mir-16 mir- 29a PGK1 mrna miscript PCR System RT-PCR RNA RNA 2 mirna mrna mirneasy Mini Micro 96 Kits RNA RNA mirna RNA mrna mrna mirna mirna mirnas RNA mirna mirneasy Serum/Plasma Kit RNA mirneasy Serum/Plasma Spike-In Control spike-in control RNA miscript PCR System FFPE RNA FFPE mirneasy FFPE Kit RNA RNA RNA DNase DNase DNase Booster Buffer RNA RT-PCR DNA mirneasy FFPE Kit miscript PCR System FFPE RNA mirna Brochure 05/12
15 Cat. no. mirneasy Mini Kit (50) Columns, plasticware, and reagents for 50 preps mirneasy Micro Kit (50) Columns, plasticware, and reagents for 50 preps mirneasy Serum/Plasma Kit (50) Columns, plasticware, and reagents for 50 preps mirneasy Serum/Plasma Spike-In Control 10 pmol C. elegans mir-39 mirna mimic spike-in control for serum/plasma samples mirneasy 96 Kit (4) Columns, plasticware, and reagents for 4 x 96 preps mirneasy FFPE Kit (50) Columns, plasticware, and reagents for 50 preps PAXgene Tissue mirna Kit (50) Columns, plasticware, and reagents for 50 preps PAXgene Blood mirna Kit (50) Columns, plasticware, and reagents for 50 preps QIAsymphony PAXgene Blood RNA Kit (96) Reagent cartridges, accessories, and buffers for 96 preps miscript II RT Kit (12) Reagents for 12 x µl cdna synthesis reactions miscript II RT Kit (50) Reagents for 50 x µl cdna synthesis reactions miscript SYBR Green PCR Kit (0) Reagents for 0 x 50 µl PCRs miscript SYBR Green PCR Kit (1000) Reagents for 1000 x 50 µl PCRs miscript PCR Starter Kit (80) Reagents for 12 x µl cdna synthesis reactions and 80 x µl PCRs miscript Primer Assay (100) mirna-specific primer for 100 x 50 µl PCRs Varies* miscript Precursor Assay (100) Precursor-specific primer pair for 100 x 50 µl PCRs Varies* mirnome miscript mirna PCR Array mirnome panels of mirna assays miscript mirna PCR Array Pathway or disease panels of mirna assays Custom miscript mirna PCR Array Custom panels of mirna assays miscript mirna Mimic 1 nmol, 5 nmol, or nmol mimic Varies* miscript mirna Inhibitor 1 nmol, 5 nmol, or nmol inhibitor (option of phosphorothioate modification) Varies* miscript Protector 5 nmol target protector Varies* * Trademarks and Disclaimers QIAGEN Handbook User Manual mirna Trademarks: QIAGEN, QIAcube, QIAsymphony, GeneGlobe, miscript, QuantiTect, Rotor-Gene, Rotor-Disc (QIAGEN Group); PAXgene (PreAnalytiX GmbH); Roche, LightCycler (Roche Group); Eppendorf, Mastercycler (Eppendorf AG); Agilent, Stratagene (Agilent Technologies); Bio-Rad, icycler, Chromo4, CFX96, DNA Engine Opticon, CFX384, iq, MyiQ (Bio-Rad Laboratories, Inc.); ABI, Applied Biosystems, StepOnePlus, ViiA, SYBR (Life Technologies Corporation); Excel (Microsoft, Inc.). RNAlater is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents /12 12 QIAGEN, all rights reserved. mirna Brochure 05/12 Sample & Assay Technologies 15
16 n n n Forefront Tower II Tel: n Fax: n techservice-jp@qiagen.com /12 Sample & Assay Technologies
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