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PCR 3 Mature mirna PCR 7 miscript mirna PCR Array C T 12 Mature mirna cdna 15 miscript mirna QC PCR Array 18 22 2 miscript mirna PCR Array 10/2011
PCR miscript II RT Kit 2 5 5x miscript HiSpec Buffer 5x miscript HiFlex BufferPathway-Focused mirna PCR Array mirnome miscript mirna PCR Array mature mirna cdna 5x miscript HiSpec Buffer mature mirnasmall noncoding RNAprecursor mirnamrna miscript HiFlex Buffer miscript PCR System Handbook mirna RNA RNA Handbook 24 2 µg RNA RNA 3 RNA Appendix B Handbook 51 RNA RNA miscript II RT Kit miscript mirna PCR Array 10/2011 3
3. RNA PCR Assay Buffer RNA * mature mirna mature mirna mirnome Pathway-Focused miscript mirna PCR Arrays mirnome miscript mirna PCR Arrays 5x miscript HiSpec Buffer 5x miscript HiSpec Buffer 125 250 ng RNA 384 4 x 96 Rotor-Disc 250 500 ng mirnome miscript mirna PCR Array * RNA RNA cdna 0.5 1 ng RNA 1 8 x 384 32 x 96 Rotor-Disc cdna 1. RNA RNase 10x miscript Nucleics Mix5x miscript HiSpec Buffer 15 25 2. 4 cdna RNA miscript Reverse Transcriptase Mix 20 2 10 4 miscript mirna PCR Array 10/2011
4. 5x miscript HiSpec Buffer 4 µl 10x miscript Nucleics Mix 2 µl RNase miscript Reverse Transcriptase Mix 2 µl RNA 3 3 * 20 µl * QIAGEN Supplementary Protocol: Purification of total RNA, including small RNAs, from serum or plasma using the mirneasy Mini Kit RNA 5 µl RNA Pathway-Focused miscript mirna PCR Array 3. RNA 4. 37 60 5. 95 5 miscript Reverse Transcriptase Mix 6. 5 RNase cdna PCR PCR cdna 20 cdna 20 miscript mirna PCR Array 10/2011 5
5. PCR cdna PCR Assay mirnome Pathway-Focused miscript mirna PCR Arrays mirnome miscript mirna PCR Arrays 200 µl RNase 20 µl mirnome miscript mirna PCR Array Rotor-Disc 1 x 384 4 x 96 Rotor-Disc90 µl RNase 20 µl 2 x 384 8 x 96 Rotor-Disc200 µl RNase 20 µl 3 x 384 12 x 96 Rotor-Disc310 µl RNase 20 µl 4 x 384 16 x 96 Rotor-Disc420 µl RNase 20 µl 6 miscript mirna PCR Array 10/2011
mature mirna PCR miscript II RT Kit miscript HiSpec Buffer cdna miscript mirna PCR Array miscript Universal Primerreverse primer QuantiTect SYBR Green PCR Master Mix miscript SYBR Green PCR Kit mature mirna PCR PCR 95 15 HotStarTaq DNA Polymerase2x QuantiTect SYBR Green PCR Master Mix miscript HiSpec Buffer cdna 20 µl cdna 6 5 cdna miscript SYBR Green PCR Kit miscript mirna PCR Array 384 10 µl96 25 µl100-well Rotor-Disc 20 µl miscript SYBR Green PCR Kit200 2 x 384 3 x 96 4 x Rotor-Disc 100 miscript SYBR Green PCR Kit1000 11 x 384 18 x 96 22 x Rotor-Disc 100 icycler iq iq5 MyiQ Well Factor Well Factor www.qiagen.co.jp Technical InformationUsing QuantiTect SYBR Green Kits on Bio-Rad cyclers miscript mirna PCR Array miscript mirna QC PCR Array cdna 15 miscript mirna PCR Array 10/2011 7
1. 2x QuantiTect SYBR Green PCR Master Mix10x miscript Universal Primer cdnarnase 15 25 2. 6Pathway-Focused miscript mirna PCR Array 7miRNome miscript mirna PCR Array PCR 6. Pathway-Focused miscript mirna PCR Array * 384 4 x 96 Formats EG 96 Formats ACDF Rotor-Disc 100 Format R 2x QuantiTect SYBR 550 µl 1,375 µl 1,100 µl Green PCR Master Mix 10x miscript Universal Primer 110 µl 275 µl 220 µl RNase 340 µl 1,000 µl 780 µl cdna 100 µl 100 µl 100 µl 1,100 µl 2,750 µl 2,200 µl * 384 10 µl 96 25 µl100-well Rotor-Disc 20 µl 1 cdna quadruplicate 4 cdna 1 384-well Pathway-Focused miscript mirna PCR Array Handbook 16 Figure 4 Mg 2+ 2x QuantiTect SYBR Green PCR Master Mix Mg 2+ 0.5 1 ng cdna 8 miscript mirna PCR Array 10/2011
7. mirnome miscript mirna PCR Array * 384 Formats EG 96 Formats ACDF Rotor-Disc 100 Format R 2x QuantiTect SYBR 2,050 µl 1,375 µl 1,100 µl Green PCR Master Mix 10x miscript Universal Primer 410 µl 275 µl 220 µl RNase 1,540 µl 1,075 µl 855 µl cdna 100 µl 25 µl 25 µl 4,100 µl 2,750 µl 2,200 µl * mirnome Rotor-Disc1 mirnome Rotor-Disc mirnome Rotor-Disc 384 10 µl 96 25 µl100-well Rotor-Disc 20 µl Mg 2+ 2x QuantiTect SYBR Green PCR Master Mix Mg 2+ 0.5 1 ng cdna 3. miscript mirna PCR Array 96 384 RT 2 PCR Array Loading Reservoircat. no. 338162 loading reservoir 4. miscript mirna QC PCR Array 384 96 Rotor-Disc 100 QIAgility 384 miscript mirna PCR Array 10 µl 96 miscript mirna PCR Array 25 µl Rotor-Disc miscript mirna PCR Array 20 µl 384 4 x 96Pathway-Focused miscript mirna PCR Arrays Pathway-Focused miscript mirna PCR Array 96 4 4 miscript mirna PCR Array 10/2011 9
384EZLoad Covers 10 384EZLoad Cover 384EZLoad Cover 1 1 10 µl ACEGIKMO 384EZLoad Cover 1 384EZLoad Cover 2 2 10 µl ACEGIKMO 384EZLoad Cover 2 384EZLoad Cover 3 3 10 µl BDFHJLNP 384EZLoad Cover 3 384EZLoad Cover 4 4 10 µl BDFHJLNP 384EZLoad Cover 4 1 Cover 1 2 Cover 2 3 Cover 3 4 Cover 4 10. E G384 Pathway-Focused miscript mirna PCR Array 4 10 µl 10 miscript mirna PCR Array 10/2011
5. Optical Thin-Wall 8-Cap StripsFormats A DOptical Adhesive Film Formats CEFG Rotor-Disc Heat-Sealing FilmFormat R miscript mirna PCR Array 6. PCR 15 25 1,000 g1 Rotor-Disc 7. 8 PCR 8. PCR PCR 3 * 15 94 30 55 15 95 HotStarTaq DNA Polymerase 30 70 40 cycles cdna * Bio-Rad models CFX96 CFX384 ramp rate 1 /s Eppendorf Mastercyler ep realplex models 22S44S Silver Thermoblock ramp rate 26 Aluminum Thermoblock ramp rate 35 Roche LightCycler 480 ramp rate 1 /s ABI PRISM 7000 30 Applied Biosystems 7300 7500 34 Roche LightCycler 480 45 8. Rotor-Disc 9. miscript mirna PCR Array 10/2011 11
miscript mirna PCR Array C T miscript mirna PCR Array miscript mirna PCR Array http://pcrdataanalysis.sabiosciences.com/mirna miscript mirna PCR Array miscript mirna PCR Array Data Analysis Excel Template Excel C T 96 384 ACDEF G 100 Rotor-Disc R 1. PCR Linear View 2 2 15 PCR Handbook 48 Appendix A Rotor-Gene Q Dynamic TubeSlope Correct Ignore First Rotor-Gene Q User Manual PCR 2. Threshold Threshold Log View 12 miscript mirna PCR Array 10/2011
Threshold Threshold Threshold PCR PCR Applied Biosystems 7500 ViiA 7 Stratagene Mx3005P Mx3000P Manual C T Threshold 0.2 0.02 Rotor-Gene Q C T 0.02 Threshold PCR 3. PCR C T 4. http://pcrdataanalysis.sabiosciences.com/mirna Excel 5. 35 N/A C T 35 33 N/A C T 33 35 33 C T negative call 6. PCR PPC C T RNA PPC Thresholds C T 19 ± 2 15 ± 2 7. mirtc C T PCR PPC mirtc PPC C T = AVG C T AVG C T 7 7 22 mirtc PPC C T = AVG C T AVG C T Pathway-Focused miscript mirna PCR Array mirnome miscript mirna PCR Array cdna Rotor-Disc mirtc 9 miscript mirna PCR Array 10/2011 13
9. mirnome miscript mirna PCR Array C T mirtc-ppc Rotor-Disc 384 96 Rotor-Disc 100 mirtc-ppc C T 1 4 4 1.1 AVG C T mirtc 1.1 AVG C T PPC 2 8 8 2.1 AVG C T mirtc 2.1 AVG C T PPC 3 12 12 2.7 AVG C T mirtc 2.7 AVG C T PPC 4 16 16 3.1 AVG C T mirtc 3.1 AVG C T PPC 8. mature mirna C T C T = C T mirna AVG C T SN1/2/3/4/5/6 snorna/snrna 6 snorna/snrna controlssn1 6 snorna/snrna miscript mirna PCR Array C T snorna/snrna snorna/snrna C T replicate replicates snorna/snrna C T 9. 2 miscript mirna PCR Array 2 mirna C T C T = C T 2 C T 1 1 2 10. 1 2 Fold-change 2 CT fold-change 1 fold upregulation fold-change 1 fold downregulation 11. Fold-changes 14 miscript mirna PCR Array 10/2011
Mature mirna cdna miscript mirna QC PCR Array miscript HiSpec Buffer cdna miscript mirna QC PCR Array 5 µl cdna miscript II RT Kit miscript HiSpec Buffer cdna miscript mirna PCR Array mirna miscript SYBR Green PCR Kit miscript mirna QC PCR Array cdna 32 cdna 1 384 miscript mirna QC PCR Array 8 cdna 1 96 miscript mirna QC PCR Array 8 cdna 1 Rotor-Disc 100 miscript mirna QC PCR Array PCR 95 15 HotStarTaq DNA Polymerase2x QuantiTect SYBR Green PCR Master Mix miscript HiSpec Buffer cdna 3 cdna 20 µl cdna 6 5 cdna miscript SYBR Green PCR Kit miscript mirna PCR Array 384 10 µl 96 25 µl100-well Rotor-Disc 20 µl icycler iqiq5 MyiQ Well Factor Well Factor www.qiagen.co.jp Technical Information: Using QuantiTect SYBR Green Kits on Bio-Rad cyclers miscript mirna PCR Array 10/2011 15
1. 2x QuantiTect SYBR Green PCR Master Mix10x miscript Universal Primer cdnarnase 15 25 2. 10 PCR 10. miscript mirna QC PCR Array * 384 Formats EG 96 Rotor-Disc 100 Formats ACDF Format R 2x QuantiTect SYBR 75 µl 175 µl 150 µl Green PCR Master Mix 10x miscript Universal Primer 15 µl 35 µl 30 µl RNase 55 µl 135 µl 115 µl cdna 5 µl 5 µl 5 µl 150 µl 350 µl 300 µl * 384 10 µl 96 25 µl100 Rotor-Disc 20 µl 1 cdna 8 cdna 1 96 miscript mirna QC PCR Array 8 cdna 1 Rotor-Disc 100 miscript mirna QC PCR Array 32 cdna 1 384 miscript mirna QC PCR Array Mg 2+ 2x QuantiTect SYBR Green PCR Master Mix Mg 2+ 3. miscript mirna QC PCR Array 4. miscript mirna QC PCR Array 384 miscript mirna PCR Array 10 µl 96 miscript mirna PCR Array 25 µl Rotor-Disc 100 miscript mirna PCR Array 20 µl 5. Optical Thin-Wall 8-Cap StripsFormats ADOptical Adhesive FilmFormats CEFG Rotor-Disc Heat-Sealing FilmFormat R miscript mirna PCR Array 16 miscript mirna PCR Array 10/2011
6. PCR 15 25 1,000 g1 Rotor-Disc 7. 11 PCR 11. PCR PCR 3 * 15 94 30 55 15 95 HotStarTaq DNA Polymerase 30 70 40 cycles cdna * Bio-Rad models CFX96 CFX384 ramp rate 1 /s Eppendorf Mastercyler ep realplex models 22S44S Silver Thermoblock ramp rate 26 Aluminum Thermoblock ramp rate 35 Roche LightCycler 480 ramp rate 1 /s ABI PRISM 7000 30 Applied Biosystems 7300 7500 34 Roche LightCycler 480 45 8. Rotor-Disc 9. miscript mirna PCR Array 10/2011 17
miscript mirna QC PCR Array miscript mirna QC PCR Array miscript mirna QC PCR Array http://pcrdataanalysis.sabiosciences.com/mirna miscript mirna PCR Array miscript mirna PCR Array Data Analysis Excel Template Excel C T 96 384 ACDEF G 100 Rotor-Disc R 1. PCR Linear View 2 2 15 PCR Handbook 48 Appendix A Rotor-Gene Q Dynamic TubeSlope Correct Ignore First Rotor-Gene Q User Manual PCR 2. Threshold Threshold Log View Threshold Threshold Threshold PCR 18 miscript mirna PCR Array 10/2011
PCR Applied Biosystems 7500 ViiA 7 Stratagene Mx3005P Mx3000P Manual C T Threshold 0.2 0.02 Rotor-Gene Q C T 0.02 Threshold PCR 3. PCR C T 4. http://pcrdataanalysis.sabiosciences.com/mirna Excel 5. 35 N/A C T 35 33 N/A C T 33 35 33 C T negative call 6. PCR PPC C T RNA PPC Thresholds C T 19 ± 2 15 ± 2 7. mirtc C T PCR PPC mirtc PPC C T = AVG C T AVG C T 7 7 22 cdna Rotor-Disc mirtc 12 miscript mirna PCR Array 10/2011 19
12. miscript mirna QC PCR Array C T mirtc-ppc cdna mirtc PPC C T Pathway-Focused miscript mirna PCR Arrays mirnome 1 x 384 4 x 96 Rotor-Disc mirnome 2 x 384 8 x 96 Rotor-Disc mirnome 3 x 384 12 x 96 Rotor-Disc mirnome 4 x 384 16 x 96 Rotor-Disc 1.5 mirtc PPC AVG C T 1.5 AVG C T 0.5 mirtc PPC AVG C T 0.5 AVG C T 1.5 mirtc PPC AVG C T 1.5 AVG C T 2.0 mirtc PPC AVG C T 2.0 AVG C T 2.5 mirtc PPC AVG C T 2.5 AVG C T 8. snorna/snrna controlssn1 6 C T small RNA snorna/snrna C T C T www.qiagen.com/mirnacontrols miscript PCR Control 20 miscript mirna PCR Array 10/2011
9. C. elegans mir-39 miscript Primer Assay C T Ce RNA Syn-cel-miR-39 miscript mirna Mimic C. elegans mir-39 miscript Primer Assay C T Threshold QIAGEN Supplementary Protocol: Purification of total RNA, including small RNAs, from serum or plasma using the mirneasy Mini Kit www.qiagen.com/mirneasyminiresources 10. cdna miscript mirna PCR Array miscript mirna PCR Array 10/2011 21
AVG C T mirtc AVG C T PPC > 7) a RNA b RNA A 260 A 280 A 260 A 230 RNase 10 mm Tris ClpH 7.5 mir- Neasy Mini Kitcat. no. 217004 RNA mirnome miscript mirna PCR Array miscript mirna QC PCR Array 14 9 20 12 PCR AVG C T PPC 2 96 384 21 100-well Rotor-Disc 17 a b HotStarTaq DNA Polymerase c RNA PCR C T PPC 96 384 19 ± 2100 Rotor-Disc 15 ± 2 2 C T PPC 95 15 RNA A 260 A 280 A 260 A 230 RNase 10 mm Tris ClpH 7.5 mirneasy Mini Kitcat. no. 217004 RNA 22 miscript mirna PCR Array 10/2011
PCR a b c RNA d RNA e RNA f RNA RNA RNA RNase cdna RT-PCR 4 3 RNA RNA RNA RNA 37 cdna miscript Reverse Transcriptase Mix miscript mirna PCR Array 10/2011 23
PCR PCR a PCR b PCR c PCR d HotStarTaq DNA Polymerase e cdna HotStarTaq DNA Polymerase f PCR g layer/filter h layer/filter cdna C T Crossing point a b a b cdna RNA 24 miscript mirna PCR Array 10/2011
Memo miscript mirna PCR Array 10/2011 25
Memo 26 miscript mirna PCR Array 10/2011
Trademarks: QIAGEN, QIAgility, HotStarTaq, QuantiTect, RNeasy, Rotor-Gene, Rotor-Disc (QIAGEN Group); ABI PRISM, Applied Biosystems (Applera Corporation or its subsidiaries); SYBR (Molecular Probes, Inc.); Bio-Rad, CFX96, CFX384, icycler, iq, MyiQ (Bio-Rad Laboratories, Inc.); Roche, LightCycler (Roche Group); Eppendorf, Mastercycler (Eppendorf AG); Stratagene (Stratagene); Excel (Microsoft, Inc.). www.qiagen.co.jp Trademarks and DisclaimersQIAGEN Handbook User Manual www.qiagen.co.jp 2012 QIAGEN, all rights reserved.
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