QuantiGene 2.0 miRNA Assay User Manual
Contents
1. Asia 81 3 6430 430 techsupport_asia affymetrix com 2 QuantiGene 2 0 miRNA Assay User Manual QuantiGene 2 0 miRNA Assay Basics The QuantiGene 2 0 miRNA Assay enables researchers to perform direct detection and quantification of miRNAs at single base resolution avoiding biases associated with miRNA or total RNA isolation cDNA synthesis and PCR amplification The assay uses validated probe sets that are highly specific for the mature miRNA and do not detect the precursor miRNA The assay is ideal for target quantification screening and validation of miRNA array results The kits provide the reagents needed to quantify specific miRNA molecules directly from Cultured cell lysates u Whole blood or PAXgene blood lysates Fresh frozen or FFPE tissue homogenates Purified miRNA or total RNA Please refer to the QuantiGene Sample Processing Kit Package Inserts for instructions on preparing cultured cell or blood lysates or tissue homogenates To prepare miRNA or total RNA follow standard laboratory methods The QuantiGene 2 0 miRNA Assay is a hybridization based assay performed on 96 well plates The assay is based on direct from lysate quantification of the miRNA target using novel oligonucleotide chemistry and probe design for specific and efficient capturing of miRNA followed by branched DNA bDNA signal amplification On the first day the sample is lysed to release the miRNAs and incubated overnight in the 92. Guidelines for Data Analysis Normalizing Gene Expression Data To normalize gene expression data Step Action 1 Calculate Average Signal Background S B for samples and normalization RNAs Note that background is defined as well containing all assay components except for target miRNA or samples 2 Calculate Normalized Signal S B of sample S B of normalization RNAs NOTE if multiple normalization RNAs are measured the geometric mean of background subtracted AVG housekeeping RNA signals may be used for data normalization Calculating Relative Fold Change of miRNA Expression Calculating miRNA Copy Number Well 11 To calculate the relative fold change of target miRNA expression in treated versus untreated samples Step Action 1 Run sample of interest using probe sets of normalization small RNA and target miRNA 2 Normalize miRNA expression data as described in Normalizing Gene Expression Data on page 10 3 Divide the normalized value for the treated sample by the normalized value for the untreated sample 4 Optional Several normalization small RNA can be run from the same sample and their Geometric Mean used for normalization purposes 5 Optional Normalization can also be performed for mRNA using the QuantiGene Assay with the same sample prepared for miRNA NOTE Given that the temperature of the two assays MRNA and miRNA will be different detectio
3. Microcentrifuge Eppendorf 541D or equivalent Microplate centrifuge that can achieve Eppendorf 5804R and rotor A 2 DWP or 240 xg equivalent Vortex mixer MLS 6 QuantiGene 2 0 miRNA Assay User Manual Table 1 6 Required Materials Not Provided Required Material Source Part Number or Model Nuclease free water MLS Yeast tRNA Invitrogen P N 15401 011 Luminescence detector with the following features u Sensitivity gt 3 x 10 21 moles of luciferase Dynamic range gt 8 logs u Well to well uniformity 596 a Cross talk lt 5 x 10 a Fluorescent detection module optional for DNA stain Ex 480 nm Em 520 nm NOTE Make sure your luminometer meets or exceeds minimum performance specifications Turner BioSystems Molecular Devices Modulus Microplate Luminometer P N 9300 001 u LMAX or equivalent the following specifications 30 200 uL 5 volume 96 or 384 channels a Angle dispensing tip u Plate stacker a Automation capable Minimal dead volume Incubator or oven with the following Affymetrix QS0700 QS0701 120V specifications QS0710 QS0711 220V Maintain a constant temperatures of 46 1 C Temperature does not vary more than 1 C a Uniform temperature throughout entire incubator 4 inch soft rubber roller or Affymetrix QS0515 QuantiGene CTC Plate Sealer Affymetrix QG0400 QuantiGene Incubator Temperature Affymetrix QS0517 Validation Kit NOT
4. 0 4 467 0 Blocking Reagent 1 0 67 0 134 0 CE 0 3 20 1 40 2 LE 0 3 20 1 40 2 Total 80 0 5362 2 10 724 4 Includes 40 overage 23 To capture target miRNA from purified RNA preparations Step Action 4 Prepare the Capture Plate A After the Capture Plate reaches room temperature minimum of 30 minutes at room temperature open the sealed foil pouch and remove the Capture Plate B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate C For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 80 uL of Working Probe Set into each assay well Avoid introducing bubbles D For 48 wells or more a Using a single channel pipette transfer Working Probe Set to a reagent reservoir IMPORTANT Do not pour or reagent shortage will occur a Using a multichannel pipette and new tips for each transfer dispense 80 uL of Working Probe Set into each assay well Avoid introducing bubbles IMPORTANT Capture Probe oligonucleotides are conjugated to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips Add sample to the Capture plate Using a new pipette tip for each transfer add 20 uL of sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix IMPORTANT Add 20 uL of nuclease free water to 3 wells for the assay background cont
5. 2 Hybridizing the 2 0 PreAmplifier Step 1 2 3 Remove all traces of Wash Buffer A Place inverted plate with a dry paper towel into the centrifuge and centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available IMPORTANT Do not exceed 240 x g for 1 minute B Proceed to the next step immediately Add 100 uL of 2 0 PreAmp Working Reagent to each well of the Capture Plate Seal the Capture Plate with a Plate Seal and incubate at 46 1 C for 60 minutes Hybridizing the 2 0 Amplifier Hybridizing the Label Probe 25 To hybridize the 2 0 Amplifier Step Action 1 Prepare 2 0 Amp Working Reagent A a 2 0 Amp then centrifuge briefly to collect the contents at the bottom of the ube B Add 11 uL of 2 0 Amp to 11 mL of Amplifier Label Probe Diluent C Invert to mix D Keep at room temperature until use 2 Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 2B 2D two more times using 300 uL well of Wash Buffer 3 Remove all traces of Wash Buffer A Place inverted plate with a dry paper towel into the centrifuge and centrifuge a
6. 5 Remove the Plate Seal place the Capture Plate in the luminometer and read Set integration read time to 0 2 seconds For best results read plate within 15 minutes NOTE Depending upon luminometer used some adjustments in integration time may be required to obtain better signal to background ratio and linearity Troubleshooting Low Assay Signal or Poor Sensitivity Table 4 1 Troubleshooting Low Assay Signal or Poor Sensitivity Probable Cause Recommended Action Number of target miRNA molecules below limit of detection Increase the sample input Verify complete cell lysis see Optimizing Sample Input for QuantiGene miRNA Assay on page 9 Signal amplification reagent incorrectly prepared Dilute 2 0 PreAmp 2 0 Amp and Label Probe in Amplifier Label Probe diluent Inappropriate hybridization temperature Hybridization reactions must be carried out at 46 1 C as directed in protocol Use a QuantiGene Incubator Temperature Validation Kit to verify and monitor the temperature Inactivation of alkaline phosphatase Do not exceed 50 C after the addition of Label Probe Do not allow the Capture Plate to stand dry for more than 5 minutes once the signal amplification and detection procedure has started Expired reagents were used Reagents are good for up to 6 months from date of receipt Luminometer does not have the required sensitivity Non Uniform Signal Across
7. Plate 26 QuantiGene 2 0 miRNA Assay User Manual Adding the 2 0 Substrate and Detecting Signal To hybridize the Label Probe Step Action 5 Seal the Capture Plate with a Plate Seal and incubate at 46 1 C for 60 minutes IMPORTANT During this incubation remove 2 0 Substrate from 4 C and allow it to warm to room temperature To add the 2 0 substrate and detecting signal Step Action 1 Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 2B 2D two more times using 300 uL well of Wash Buffer 2 Remove all traces of Wash Buffer A Place inverted plate with a dry paper towel into the centrifuge and centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available IMPORTANT Do not exceed 240 x g for 1 minute IMPORTANT Ensure that 2 0 Substrate is at room temperature before use and that the luminometer is ready for use B Proceed to the next step immediately 3 Add 100 uL of 2 0 Substrate to each well of the Capture Plate Seal the Capture Plate with a Plate Seal and incubate at room temperature for 5 minutes
8. conjugated to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips Add sample to the Capture plate Using a new pipette tip for each transfer add 80 uL of sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix IMPORTANT Add 80 uL of Diluted Lysis Mixture 1 volume Lysis Mixture plus 2 volumes nuclease free water to 3 wells for the assay background controls Run background wells for each Probe Set used Bind target miRNA A Place an adhesive Plate Seal squarely on the plate and seal tightly IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 46 1 C incubator to begin the overnight 16 20 hour hybridization IMPORTANT Temperature must be 46 1 C for miRNA assay Verify temperature using a QuantiGene Incubator Temperature Validation Kit To capture target miRNA from fresh frozen or FFPE tissue homogenates Step Action 1 Prepare reagents Probe Set s and Blocking Reagent Thaw vortex briefly to mix then br
9. miR 93 in Corn Husk Lysate no lysate g with corn husk lysate 10 000 100 000 1 000 000 miRNA copies per well 10 000 000 16 QuantiGene 2 0 miRNA Assay User Manual QuantiGene 2 0 miRNA Assay Procedure Assay Workflow Table 3 1 Step Tasks 1 Prepare samples 2 Capture miRNA a Dilute samples a Prepare Working Probes Sets Dispense Working Probe Sets samples and controls into Capture Plate Hybridize overnight 3 Amplify and detect signal Wash away unbound material Sequentially hybridize 2 0 PreAmp Amp and Label Probe a Add 2 0 Substrate incubate and read signal For a procedure refer to Appropriate QuantiGene Sample Processing Kit for preparing cultured cell lysate blood lysates and tissue homogenates Follow standard laboratory methods for purification of RNA Use samples immediately or store at 80 C until ready to use Capturing Target miRNA Refer to the appropriate procedure for your sample type Cultured cell or blood lysates Fresh frozen or FFPE tissue homogenates Total RNA or purified miRNA Capturing Target miRNA from Cultured Cell or To capture target miRNA from cultured cell or blood lysates Whole Blood Step Action PAXgene Blood 1 Prepare reagents Probe Set s and Blocking Reagent Thaw vortex briefly to mix then briefly centrifuge to collect contents at the bottom of the tubes Keep on ice until use a Cult
10. protein containing preservative Lysis Mixture Aqueous buffered solution 15 30 C containing a preservative Plate Seals Adhesive backed foil seal 15 30 C Wash Buffer Component 1 Aqueous solution 15 30 C Wash Comp 1 Wash Buffer Component 2 Aqueous buffered solution 15 30 C Wash Comp 2 2Lumigen APS 5 QuantiGene 2 0 miRNA Assay Accessory Reagents Optional Upstream Cell Viability Assay Required Materials Not Provided In addition to QuantiGene 2 0 Assay Kits two accessory reagents are required to perform QuantiGene 2 0 miRNA assays Table 1 4 QuantiGene 2 0 miRNA Assay Accessory Components Accessory Reagent Description Required QuantiGene Sample Processing Kit Contains reagents and instructions for processing different sample types cultured cells whole blood PAXgene blood fresh or frozen tissues or FFPE samples Specify sample type when ordering not required for purified RNA Required QuantiGene miRNA target specific Probe Set s Customer specified miRNA target s Each Probe Set contains 1 CE and 1 LE Optional QuantiGene miRNA Probe Set s for Normalization Customer specified small RNA s or miRNA targets suitable for normalizing These targets should have characteristics of a traditional housekeeping gene Target is stably expressed under all experimental conditions evaluated Each Probe Set contains 1 CE and 1 LE Optional miRNA Positive Co
11. the best performance and use that amount of cells or tissue for future experiments Incomplete or poor lysis will produce high assay CV poor linearity and poor LOD To determine the optimal lysis method for a sample type Following the procedure for determining optimal lysis test different lysis methods for example Tissue lyser or liquid nitrogen for plant tissues Optimizing After you have determined the optimal lysis conditions for sample preparation use the Sample Input for following guidelines to determine the optimal sample amount well to use for the QuantiGene miRNA QuantiGene 2 0 miRNA assay Assay Resulting signal from the sample is above the LOQ Amount of sample is high enough to compensate for sample loading error For example if the amount of loaded sample can deviate more than 4 times then increase the sample input by 4 to ensure detection 10 QuantiGene 2 0 miRNA Assay User Manual Assay Replicates Recommended Assay Controls Ifthe amount of sample is not limiting use an input that has a signal background ratio of at least 3 fold Background is defined as signal from a sample well without sample input Run all assay samples with a minimum of duplicates and ideally triplicates Technical replicates are used to calculate assay precision or CV All experiments should have the following controls Assay Background Control A sample well that contains all assay components except
12. the Plate Only use luminometers that meet or exceed the minimum performance specifications see Optimizing Sample Input for QuantiGene miRNA Assay on page 9 Table 4 2 Troubleshooting Non Uniform Signal Across the Plate Probable Cause Recommended Action Temperature gradients within the incubator Verify that the incubator maintains a constant even temperature throughout the incubator Avoid opening and closing the incubator door during hybridization steps Temperature gradients on Capture Plate at time of reading Read plate at room temperature If luminometer has heating capability ensure that this function is turned off and indicates room temperature Incomplete sealing during overnight hybridization Use the CTC Plate Sealer for robust plate sealing Affymetrix P N QG0400 Ensure numbers and letters are clearly visible from under the foil seal Verify that the supplied plate seal was used Capture Plates exposed to moisture prior to the assay Allow the Capture Plate to come to room temperature for 30 minutes before opening the sealed foil pouch to avoid condensation Variable salt concentrations Hybridization is affected by salt When diluting samples always use the appropriate diluent 28 QuantiGene 2 0 miRNA Assay User Manual High Background Signal Well To Well Variation Table 4 3 Troubleshooting High Background Signal Probable Cause Recommended Acti
13. 6 well plates with the target specific probe sets Capture Extenders CEs and Label Extenders LEs On the second day the signal amplification tree is built via sequential hybridization of PreAmplifier PreAmp Amplifier Amp and Alkaline phosphatase label probe AP LP The signal is detected by adding chemiluminescent substrate and using a microplate luminometer for the read out How it Works Purified miRNA or small RNA Whole blood O Animal A tissues Cultured cells FFPE sections Lyse amp Go Assay Specifications Capture Probe PreAmplifier Chemiluminescent Substrate Capture Extender Amplifier f Label Probe Q Zo Label Extender for signal Read on a qu amplification Luminometer Lumigen APS 5Capture Table 1 2 Assay Specifications Component Description Assay Format 96 well plate Sample Types u Purified miRNA u Purified Total RNA Whole Blood PAXgene Blood Lysates u Cell Culture Lysates a Plant Animal and Insect Tissue homogenates Limit of Detection Greater than Average Background 3 000 60 000 copies well Signal 3SD Linear range Entire range must meet accuracy of fold 3 Log change requirement Linear regression coefficient R2 greater than 0 98 of Spike Recovery a 100 20 range 80 120 Accuracy of Fold Change of Observed Expected Fold 100 20 range 80 120 Change Inter wells Precisi
14. 73 0 6576 7 720 907 copies 8 000 cells Therefore copy number cell 7 720 907 copies 8 000 cells 965 copies cell Spike recovery experiments determine the assay s ability to capture and detect a specific miRNA target in a complex matrices of lysates consisting of proteins lipids carbohydrates and nucleic acids The QuantiGene 2 0 miRNA Assay must meet the spike recovery of 100 20 or a range of 80 120 To calculate spike recovery Step Action 1 Prepare serial dilutions of miRNA Positive Control from Affymetrix in lysates or homogenates for spiked samples Include 0 miRNA as background a If preparing spiked samples in lysates dilute Affymetrix miRNA Positive Control in the lysate of interest with a final volume of 80 ul assay well a If preparing spiked samples in homogenates dilute Affymetrix miRNA Positive Control in the homogenate of interest with a final volume of 40 ul assay well 2 Prepare serial dilutions of miRNA Positive Control from Affymetrix in assay buffer for unspiked samples Include 0 miRNA as background a If preparing unspiked samples for lysates dilute Affymetrix miRNA Positive Control in Diluted Lysis Mixture plus 2 5 ng ul of Yeast tRNA Invitrogen 15401 011 with a final volume of 80 ul assay well a If preparing unspiked samples for homogenates dilute Affymetrix miRNA Positive Control in Homogenizing Solution plus 5 ng ul of Yeast tRNA Invitrogen 15401 011 with a final vo
15. Capture Plate after washing steps is not necessary 24 QuantiGene 2 0 miRNA Assay User Manual To prepare the wash buffer Action Add to a 500 mL graduated cylinder in this order A 400 mL double distilled water ddH O B 1 5 mL Wash Comp 1 C 2 5 mL Wash Comp 2 D Bring volume to 500 mL with ddH O IMPORTANT Scale preparation according to the number of plates to be processed 500 mL is sufficient for processing one Capture Plate Transfer to a 500 mL bottle and invert to mix Do not store unused Wash Buffer Make Wash Buffer fresh daily To hybridize the 2 0 PreAmplifier Action Prepare PreAmp Working Reagent A Thaw 2 0 PreAmp then centrifuge briefly to collect the contents at the bottom of the tube B Add 11 uL of 2 0 PreAmp to 11 mL of Amplifier Label Probe Diluent C Invert to mix D Keep at room temperature until use Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 2B 2D two more times using 300 uL well of Wash Buffer IMPORTANT For recommendations on automated plate washing see Alternative Plate Washing Method on page 31 Preparing Wash Buffer Step 1
16. E Highly recommended for temperature validation of incubator every 6 months Optional Plate washer that meets or exceeds _ BioTek ELx 405 model with high throughput pump option Assay Terminology and Guidelines for Data Analysis Assay Terminology Assay A sample well contains all assay components except for target miRNA or sample The Background background control is used to determine Limit of Detection LOD Replicates Technical replicates are replicate assays from a single sample For example a cell lysate that is divided into several portions and each portion run in the same assay Biological replicates are replicate assays from biologically equivalent samples For example cells grown in different wells that are subjected to the same treatment lysed independently then run as distinct samples in the assay NOTE We recommend running 3 technical replicates of each distinct biological sample Assay The Coefficient of Variation CV is a measure of assay precision QuantiGene 2 0 miRNA Precision Assay CVs are typically less than 10 for technical replicates To determine the assay CV Step Action 1 Run technical replicates n 3 of each sample 2 Calculate the average signal AVG of technical replicates from each target miRNA 3 Calculate the standard deviation SD of signals from technical replicates for each target miRNA 4 Calculate the CV CV SD AVG 100 Relative The output sig
17. KK KI KK KK KK KK KK k 29 Alternative Plate Washing Method 31 Automated Washing Procedure 31 Capture Plate Dimensions 33 About Capture Plate Dimensions 33 Blank Plate Map 2 xa adu kb a kk kK kK kK KK KK Rd KK KK KK KK KK KK KK aed dd 35 Introduction About This User Manual Who This Manual is For What this Manual Covers This manual is for anyone who has purchased QuantiGene 2 0 Assay Kits to perform the QuantiGene 2 0 miRNA Assay for any of the following sample types Cultured cells Whole blood PAXgene blood Fresh frozen or formalin fixed paraffin embedded FFPE tissues a Total RNA or purified miRNA This manual provides Background information about QuantiGene 2 0 miRNA Assay and how it works Experimental design and data analysis QuantiGene 2 0 miRNA Assay Troubleshooting Contacting Affymetrix Technical Help For technical support contact the appropriate resource provided below based on your geographical location For an updated list of FAQs and product support literature visit our website at www affymetrix com panomics Table 1 1 Contacting Affymetrix Location Contact Information North America 1 877 726 6642 option 1 then option 2 pqbhelp affymetrix com Europe 44 1 628 552550 techsupport_europe affymetrix com
18. Target MIRNA Ak kk kk kk kk kK kK KK KK KK KK K KK KK K K KK KI KK KK KI KK KK KK KK ee 17 Capturing Target miRNA from Cultured Cell or Whole Blood PAXgene Blood 17 Capturing Target miRNA from Fresh Frozen or FFPE Tissue Homogenates 19 Capturing Target miRNA from Total RNA or Purified miRNA 21 Signal Amplification and Detection kk kk kk kk kk kK KK KK KI KK KK KK K KK KK KK KK KIIR KK K 23 Preparing Wash Buffer o draco doa a reeset KK KK KK oe K KK KK KI E des KK Dose 24 iv QuantiGene 2 0 Reagent System User Manual Chapter 4 Appendix A Appendix B Appendix C Hybridizing the 2 0 PreAmplifier isses er KK KK KI KIRI K KI KI KI KIRI K K KI KI KK KK 24 Hybridizing the 2 0 Amplifier kk kk kk kk 6555 KK KK KK KK KI KI KI K K KI KI KI KI KI K rmm KI KK KK 25 Hybridizing the Label Probe 4444444 KK KK KK KK RR hh mms 25 Adding the 2 0 Substrate and Detecting Signal 26 Troubleshooting cus sci a ss kak RENE Gi KA aud phase E RO ERE ae 27 Low Assay Signal or Poor Sensitivity ii sies sare khe a KK KK IK K K awed 27 Non Uniform Signal Across the Plate 27 High Background SIgnal sas 03 6 idea n ay dcr ed 4426 kun 28 Well To Well Variation re kk kk eed ERG KK Reo KK KK KI KK KK KI KIR Qo KK KH 28 Day To Day Variation kk kk kk kk kk kK KK KK KK KK KK KK KI KK KK
19. XWe Affymetrix User Manual QuantiGene 2 0 miRNA Assay For research use only Not for use in diagnostic procedures Trademarks E Affymetrix and AMG are trademarks of Affymetrix Inc QuantiGene is a registered trademark exclusively licensed to Affymetrix Inc Affymetrix Axiom Command Console DMET GeneAtlas GeneChipG GeneChip compatible GeneTitanG Genotyping Console myDesign NetAffx OncoScan and Powered by Affymetrix are trademarks or registered trademarks of Affymetrix Inc Luminex and xMAPO are registered trademarks of Luminex Corp All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a nonexclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered
20. ber of assays to be run and include 4096 overage NOTE Include 3 wells for assay background controls Preparation of Working Probe Sets Reagent 1 Well pL 48 Wells pL 96 Wells uL Nuclease free 25 1 1 682 0 3 363 0 Water Lysis Mixture 33 3 2 233 0 4 467 0 Blocking Reagent 1 0 67 0 134 0 CE 0 3 20 1 40 2 LE 0 3 20 1 40 2 Total 60 0 4 022 2 8 044 4 a Includes 40 overage Capturing Target miRNA from Total RNA or Purified miRNA 21 To capture target miRNA from fresh frozen or FFPE tissue homogenates Step Action 4 Prepare the Capture Plate A After the Capture Plate reaches room temperature minimum of 30 minutes at room temperature open the sealed foil pouch and remove the Capture Plate B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate C For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 60 uL of Working Probe Set into each assay well Avoid introducing bubbles D For 48 wells or more a Using a single channel pipette transfer Working Probe Set to a 25 mL divided reagent reservoir IMPORTANT Do not pour or reagent shortage will occur a Using a multichannel pipette and new tips for each transfer dispense 60 uL of Working Probe Set into each assay well Avoid introducing bubbles IMPORTANT Capture Probe oligonucleotides are conjuga
21. by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 5 945 334 6 140 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 6 955 915 and D430 024 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 and 6 054 270 Software products may be covered by one or more of the following patents U S Patent Nos 6 090 555 6 611 767 6 687 692 6 829 376 7 130 458 7 451 047 and other U S or foreign patents Fluidics stations Products may be covered by U S Patent Nos 6 114 122 6 287 850 6 391 623 6 422 249 and other U S or foreign patents Scanners Products may be covered by one or more of the following patents U S Patent Nos 6 141 096 6 262 838 6 294 327 6 403 320 6 407 858 6 597 000 7 406 391 and other U S or foreign patents Hybridization ovens Products may be covered by one or more of the following patents U S Patent Nos 6 050 719 6 386 749 6 705 754 and other U S or foreign patents Citing QuantiGene 2 0 miRNA Assay in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene 2 0 miRNA Assay Disclaimer Affymetrix Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure a
22. ccuracy Affymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2011 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Introduction HHHH HH HN 1 About This User Moan al 1 kiyak a Maml uses Oe Et ee Ee a E ee s J 1 Who This Manual is Fors sse a a a salek einge dere al Rue s RR REG ras 1 What this Manual Covers 1 Contacting AffymetfiX A ss ls a A aa nnn 0480 ee 8 a d dide d Enid Shin ATRE EG se dia dik aa 1 Technical Help e sual A Oe k n L AL WA ead Wawa SS e ee al 1 QuantiGene 2 0 miRNA Assay Basics 2 HOwit VVOTKS 2o gt bkUCkkx2k k K5K 5rprp p w r BBT TIRI IR II oo a aRaa r w eeeeeeeeeebe ene bbeaaa 3 Assay Specifications a xd trr W da hn 3 Required Valerial5 s 3 5 lt lt ean ae Tex an K na aA bn e ee A et iet 4 QuantiGene 2 0 Assay Kit Components 4 QuantiGene 2 0 miRNA Assay Accessory Reagents 5 Optional Upstream Cell Viability Assay 5 Required Materials Not Provided 5 Assay Terminology and Guidelines for Data Analysis 7 Assay Terminology o celis ER
23. cross talk lt 0 001 Variable salt concentrations Hybridization is affected by salt When diluting samples always use the appropriate diluent Inaccurate pipetting Only use calibrated precision pipettes Affix tips securely Use a new tip for each transfer L L L Pipet slowly and carefully avoiding bubbles Non homogenous samples Warm samples to 37 C to dissolve any precipitates and vortex briefly before use Samples too viscous to pipet accurately Dilute samples 1 2 in the appropriate diluent before use Day To Day Variation 29 Table 4 5 Troubleshooting High Inter Plate CVs Probable Cause Recommended Action Variable incubation temperatures Keep incubation temperatures consistent Variable incubation times Keep incubation times consistent especially for incubation with 2 0 Substrate Non constant time between addition of 2 0 Substrate and plate read Make sure that time between addition of 2 0 Substrate and plate read is consistent 30 QuantiGene 2 0 miRNA Assay User Manual Alternative Plate Washing Method Automated Washing Procedure NOTE Automated washing of plates might require the purchase of additional Wash Buffer Program the BIO TEK ELx405R washer with settings for the dispense program D3 and the wash programs 44 and 45 Link the dispense program D3 to the wash programs 44 and 45 to yield Link 1 and 2 respectively Use L
24. de the Capture Plate dimensions to enable you to setup and validate alternative automated plate washers NOTE The Capture Plate construction adheres to the Society for Biomolecular screening standards Figure B 1 oU 1 ERO bottom View 10 8 mm Well depth Top View 14 3 mm Plate height 14 0 mm Well A1 x offset D 11 2 mm Well A1 y offset D 9 0 mm Well to well offset B 85 5 mm Plate width 127 8 mm Plate length 34 QuantiGene 2 0 miRNA Assay User Manual Blank Plate Map 36 QuantiGene 2 0 miRNA Assay User Manual
25. dl kull bb d 0 i ob Shed beter Ob keleha eb b r rei 7 Assay Background aoii oda Pnd Q Ur Ah le he n ak Ad Oe Wun d n Ae 7 elel S chine Ra D patient heehee DS 1A MM HE 7 ASSAY PECCISIOMI i s ed oss tectam a a De e eb E mn ES Ratt oe 7 Relative Luminescent Unit RLU 1 0 0 0 a a kk kk KK KI KIRI KII eee 7 Assay Limit of Detection LOD 7 Limit of Quantification LOQ silka kak a ada se 7 Assay Linearity Accuracy of Fold Change 8 Guidelines for Assay Optimization and Assay Design 9 OvervieW l l HHH E EH eee 9 Optimizing Lysis Conditions 9 Optimizing Sample Input for QuantiGene miRNA Assay 9 Assay Replicates NH HHHHRBE eet eee ee 10 Recommended Assay Controls 10 Guidelines for Data Analysis 10 Normalizing Gene Expression Data 10 Calculating Relative Fold Change of miRNA Expression 11 Calculating miRNA Copy Number Well 11 wel Ce n nn Penn eres tae se rene de Sak aC TR RN TUR aoe 13 QuantiGene 2 0 miRNA Assay Procedure 17 Assay WOKO en y WA more S tts are eas Bh nd CA ae Uns tom ee ee ne 17 Capturing
26. for the target miRNA The background control is also used to determine the limit of detection LOD Data below the LOD should not be used for quantification u miRNA Positive Control miRNA positive controls are provided by Affymetrix to be used as the assay positive control It is also provided to determine the absolute number of miRNA copies sample well The miRNA copy number can be determined by running an 8 point standard curve and linear curve fitting see data analysis guideline below Using the absolute copy number improves data accuracy and precision for inter day and inter site experiments and is critical for biomarker discovery and clinical research u Normalization small RNA s Considerations for the selection of the normalization gene to determine relative fold changes or normalize gene expression data across samples or experiments are listed below Genes are stably expressed under all experimental conditions evaluated Relative luminescent signal should be similar to test samples so no dilution factor is required In situations where samples are limited select the normalization gene that is highly expressed and which would require only small amount of sample for detection Examples of recommended small RNAs include Human SNORD43 SNORD44 SNORD48 Mouse Snord68 Z38 and Z39 Rat Z38 Z39 and U6 For a list of available Probe Sets for normalization small RNAs please go to www affymetrix com panomics
27. iefly centrifuge to collect contents at the bottom of the tubes Keep on ice until use a Tissue homogenates If previously frozen thaw at room temperature followed by incubation at 37 C for 15 30 minutes Vortex briefly then leave at room temperature until use a Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minutes followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 20 QuantiGene 2 0 miRNA Assay User Manual To capture target miRNA from fresh frozen or FFPE tissue homogenates Step Action 2 Determine sample input Estimate sample input based on this chart and the LOD of miRNA target specific probe set from the package insert Recommended Sample Input based on LOD 3 000 Copies of miRNA Probe Set miRNA copies per cell Tissue Homogenate pL 10 40 100 40 gt 1 000 4 May not have sensitivity required If appropriate based on the expression level of target miRNA of interest dilute tissue homogenates with Homogenizing Solution so that the desired amount of sample present in a volume of 40 ul assay well Prepare Working Probe Set Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed Scale according to the num
28. ink 1 to wash the Capture Plates after the overnight hybridization of the sample with the target specific Probe Set after the 2 0 Pre Amplifier hybridization and the 2 0 Amplifier hybridization Use Link 2 to wash the Capture Plates after the Label Probe hybridization Table A 1 ELx405R Washer Settings Parameter Program D3 44 45 Method Number of cycles 3 5 Soak Shake Yes Yes Soak duration 10 seconds 10 seconds Shake before soak No No Prime after soak No No Prime volume Prime flow rate Dispense Dispense volume 290 395 395 Dispense flow rate 5 5 5 Dispense height 115 115 115 Horizontal dispense position 10 10 10 Horizontal Y dispense position 0 0 0 Bottom wash first No No No Bottom dispense volume Bottom flow rate Bottom dispense height Bottom dispense position Prime No No No Prime volume Prime flow rate 32 QuantiGene 2 0 miRNA Assay User Manual Table A 1 ELx405R Washer Settings Parameter Program D3 44 45 Aspiration Aspirate height 32 32 Horizontal aspirate position 45 45 Horizontal Y aspirate position 0 0 Aspirate rate 5 5 Aspirate delay 0 0 Crosswise aspirate No No Crosswise aspirate on Crosswise height Crosswise horizontal position Final aspirate Yes Yes Final aspirate delay 2 seconds 2 seconds Capture Plate Dimensions About Capture Plate Dimensions We provi
29. lume of 40 ul assay well 3 Run assay acquire signal and determine background subtracted signal for spiked and unspiked miRNA 14 QuantiGene 2 0 miRNA Assay User Manual Step Action 4 To determine spike recovery spiked S B unspiked S B x 100 Must be within 80 120 S B signal background 0 miRNA NOTE Any endogenous miRNA may interfere with the spike recovery results We have designed a negative miRNA sequence derived from bacterial gene dapB Affymetrix P N SMC 10180 to perform spike recovery in various animal and plant lysates We have also demonstrated the spike recovery of human miR 93 in Corn Husk lysate See data below Spike recovery for dapB miRNA from Corn Husk lysate Spike Recovery dapB miRNA in Corn Husk Lysate 1000 e no lysate with corn husk lysate o 100 z 2 o i o5 10 c T z a0 a 1 0 1 10 000 100 000 1 000 000 10 000 000 100 000 000 miRNA copies per well Spike recovery for dapB miRNA from Stomach Tissue lysate Spike Recovery dapB miRNA in Stomach Tissue Lysate 1000 e no lysate g with stomach tissue lysate Uo 100 c 2 o0 10 c T e o0 o 1 0 1 10 000 100 000 1 000 000 10 000 000 100 000 000 miRNA copies per well 15 Step Action Spike recovery for miR 93 miRNA from Corn Husk lysate Signal Background 1000 100 10 Spike Recovery
30. n of mRNA and miRNA cannot be performed on the same plate therefore plan on using separate plates for each Determination of miRNA copy number can be achieved accurately and easily using the QuantiGene 2 0 miRNA assay In practice spike recovery experiments are used to assure specific detection of miRNA copy number in complex sample matrices and cell lysates The QuantiGene 2 0 miRNA assay has an excellent spike recovery of 100 20 Determining absolute copy number compared to relative fold changes provides the ability to compare inter day inter lab and inter group experiments This makes the QuantiGene 2 0 miRNA assay practical for both clinical and biomarker translational research An example is provided to demonstrate how to determine copy number of miR 145 in 8 000 HeLa cells Step Action 1 Use Affymetrix miR 145 Positive Control at 250 pMol 150 million copies ul to create an 8 point standard curve in triplicate a Dilute miR 145 Positive Control in nuclease free water plus 10 ng ul Yeast tRNA Invitrogen 15401 011 with a final volume of 20 ul assay well Include one background point of 0 copies miR 145 Positive Control Prepare the lowest dilution point at 2 times above the LOD of miRNA probe set refer to product insert For miR 145 the probe set LOD is 30 000 copies so that the lowest dilution point would be 60 000 copies Run standard curve dilutions and sample lysates 12 QuantiGe
31. nal on the luminometer Luminescent Unit RLU Assay Limit of The LOD is the signal above the background plus 3 standard deviations of the background Detection LOD To calculate assay limit of detection for each target miRNA LOD AVG RLU of assay background control wells 3X SD of assay background signals Assay signals below LOD should not be used for quantifications Limit of LOQ is the lowest RLU that exhibits acceptable accuracy of fold change see Assay Quantification Linearity Accuracy of Fold Change below LOQ 8 QuantiGene 2 0 miRNA Assay User Manual Assay Linearity Accuracy of Fold Change Assay linearity is defined as all dilutions that exhibit an accuracy of fold change between 80 and 120 Assay must exhibit 3 log linearity based on 80 120 accuracy of fold change Typically the R linear regression coefficient value is greater than 0 98 To determine assay linearity Step Action 1 Run a dilution series of your sample 2 Subtract the AVG assay background signal from the AVG signal of technical replicates for each target miRNA 3 Calculate the ratio of background subtracted AVG RLU from sequential sample dilutions for each target miRNA 80 120 NOTE Quantifiable signals are those signals within the assay s linear range Ratio of Background Subtracted AVG RLU for Each Target miRNA 3 fold serial Signal Observed Expected Obs dilu
32. ne 2 0 miRNA Assay User Manual Step Action Determine signal background for standard curve dilutions and samples Background is assay wells of 0 copies miR 145 Standard Curve HeLa Lysate 8 000 cells miR 145 LOD AVG Signal AVG AVG 30 000 copies Signal Signal Background Background 0 38 227 4 819 496 60 000 83 453 45 226 120 000 127 236 89 009 600 000 478 620 440 393 3 000 000 2 164 208 2 125 981 15 000 000 9 767 725 9 729 498 30 000 000 19 289 475 19 251 248 60 000 000 39 714 450 39 676 223 Plot a graph of the signal background y axis and miRNA copy number x axis Standard curve for miR 145 Positive Control 45 000 000 40 000 000 35 000 000 30 000 000 25 000 000 20 000 000 15 000 000 Signal Background 10 000 000 5 000 000 0 miR 145 Standard Curve Linear Curve Fitting y 0 6576x 25773 R 0 9998 20 000 000 40 000 000 miR 145 Copies Well 60 000 000 of Spike Recovery 13 Step Action Using linear regression curve fitting Microsoft Excel or other program to determine the linear equation and regression coefficient R2 Note that the R must be equal or greater than 0 96 To calculate miR 145 copy number cell using linear curve fitting equation Y 0 6576X 25773 X Y 25773 0 6576 If Y value 4 819 496 signal background Then X value 4 819 496 257
33. ntrol 250 pMol 150 million copies pl Synthetic RNA oligo with exactly the same sequence as the target miRNA can be used as an assay positive control or for accurate determination of miRNA copies in samples For ordering information please visit our website at www affymetrix com panomics The QuantiGene Cell Viability Reagent provides a simple reliable and sensitive means for quantifying cell proliferation and viability upstream of QuantiGene QuantiGene Plex and QuantiGene ViewRNA Plate Based assays This homogeneous assay utilizes the non fluorescent redox dye resazurin which is converted by metabolically active cells to resorufin a highly fluorescent product Ex 530 570 nm Em 590 620 nm Table 1 5 QuantiGene Cell Viability Reagent Component Description Storage QuantiGene Cell Viability Reagent Non fluorescent redox dye in aqueous 20 C solution Other materials required to perform the QuantiGene 2 0 miRNA Assay that are not included in the assay kit are listed here Table 1 6 Required Materials Not Provided Required Material Source Part Number or Model Adjustable single and multi channel precision pipettes for dispensing 1 20 uL 20 200 uL and 200 1000 uL Major laboratory supplier MLS Reagent reservoirs 25 mL capacity 100 mL capacity VistaLab Technologies a P N 3054 1002 or equivalent a Corning Costar P N CLS 4873 or equivalent
34. on Residual Wash Buffer Ensure that the plate wash method completely removes all residual Wash Buffer prior to moving to the next step in the procedure Incorrect temperature in the incubator Verify incubation temperatures using a QuantiGene Incubator Temperature Validation Kit Expired reagents were used Reagents are good for 6 months from the date of receipt Capture Plate sat at room temperature longer than 10 minutes after the addition of sample Do not let the Capture Plate sit at room temperature for longer than 10 minutes after the addition of the overnight hybridization mixture Capture Plate sat at room temperature for longer than 10 minutes before washing 2nd day Wash the Capture Plate within 10 minutes after removal from the incubator Cross talk between wells Only use Luminometers with cross talk lt 0 001 Reduce integration time on Luminometer to minimize cross talk Move high expressing samples away from background wells Table 4 4 Troubleshooting Assay CVs Probable Cause Recommended Action Residual Wash Buffer Ensure that the plate wash method completely removes all residual Wash Buffer prior to moving to the next step in the procedure Scratching of the capture well surface Minimize contact with the Capture Plate well surfaces during all addition and washing steps Cross talk among neighboring wells during reading Only use luminometers with
35. on SD of 3 replicates u t 1096 Inter plates Precision SD of 3 replicates u t 1596 4 QuantiGene 2 0 miRNA Assay User Manual Required Materials The QuantiGene 2 0 miRNA Assay is a modular kit Each module below is sold separately in multiple sizes QuantiGene 2 0 Assay Kit QuantiGene Sample Processing Kit not required for purified RNA samples QuantiGene miRNA Probe Set s QuantiGene 2 0 The components of the QuantiGene 2 0 Assay Kit and their recommended storage Assay Kit conditions are listed below The QuantiGene 2 0 Assay Kit is available in 4 sizes Refer to Components the package insert for quantities of individual components supplied Kits have a shelf life of 6 months from date of receipt when stored as recommended and contain the following components Table 1 3 QuantiGene 2 0 Assay Kit Components and Their Storage Conditions Component Description Storage 2 0 PreAmplifier PreAmp 1 DNA in aqueous buffered 20 C solution 2 0 Amplifier Amp 1 DNA in aqueous buffered 20 C solution Blocking Reagent Aqueous buffered solution 20 C containing a preservative Capture Plate 96 well polystyrene plate 2 8 C coated with capture probes Label Probe Oligonucleotide alkaline 2 8 C phosphatase conjugate in aqueous buffered solution 2 0 Substrate Chemiluminescent substrate 2 8 C Amplifier Label Probe Diluent Aqueous buffered solution with 15 30 C a
36. owing reagents in the order listed Scale according to the number of assays to be run NOTE include 3 wells for assay background controls Preparation of Working Probe Sets Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free 11 7 786 0 1 573 0 Water Lysis Mixture 6 7 447 893 0 Blocking Reagent 1 0 67 0 134 0 CE 0 3 20 1 40 2 LE 0 3 20 1 40 2 Total 20 0 1 340 2 2 680 4 a Includes 40 overage Capturing Target miRNA from Fresh Frozen or FFPE Tissue Homogenates 19 To capture target miRNA from cultured cell or blood lysates Step Action 4 Prepare the Capture Plate A After the Capture Plate reaches room temperature minimum of 30 minutes at room temperature open the sealed foil pouch and remove the Capture Plate B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate C For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 20 uL of Working Probe Set into each assay well Avoid introducing bubbles D For 48 wells or more a Using a single channel pipette transfer Working Probe Set to a 25 mL divided reagent reservoir NOTE Do not pour or reagent shortage will occur a Using a multichannel pipette and new tips for each transfer dispense 20 uL of Working Probe Set into each assay well Avoid introducing bubbles IMPORTANT Capture Probe oligonucleotides are
37. rols Run background wells for each Probe Set used Bind target miRNA A Place an adhesive Plate Seal squarely on the plate and seal tightly IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 46 1 C incubator to begin the overnight 16 20 hour hybridization IMPORTANT Temperature must be 46 1 C for miRNA assay Verify temperature using a QuantiGene Incubator Temperature Validation Kit Signal Amplification and Detection These instructions are for processing a single Capture Plate using multichannel pipettes and reagent reservoirs To process more than one Capture Plate scale reagents accordingly If using a 50 plate kit scale reagent preparations for a minimum of 10 plates per run or reagent shortages will occur IMPORTANT Do not let the Capture Plate s stand dry for more than 5 minutes at any point in this procedure IMPORTANT Incubation temperatures must be 46 1 C Verify temperatures using a QuantiGene Incubator Temperature Validation Kit IMPORTANT If using a recommended plate washer centrifugation of the
38. rozen thaw on ice a Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minutes followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 22 QuantiGene 2 0 miRNA Assay User Manual To capture target miRNA from purified RNA preparations Step Action 2 Determine sample input Estimate sample input based on this chart and the LOD of miRNA target specific probe set from the package insert Recommended Sample Input based on LOD 3 000 Copies of miRNA Probe Set miRNA copies per cell Total RNA ng lt 10 100 100 10 gt 1 000 1 2100 ng of RNA is approximately equivalent to 5 000 cells Dilute RNA in nuclease free water so that the desired amount of RNA is present in a volume of 20 uL assay well based on expression level of target miRNA of interest Prepare Working Probe Set Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 4096 overage IMPORTANT include 3 wells for assay background controls Preparation of Working Probe Sets Reagent 1 Well pL 48 Wells pL 96 Wells uL Nuclease free 45 1 3 022 0 6 043 0 Water Lysis Mixture 33 3 2 233
39. t 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available IMPORTANT Do not exceed 240 x g for 1 minute B Proceed to the next step immediately Add 100 uL of 2 0 Amp Working Reagent to each well of the Capture Plate Seal the Capture Plate with a Plate Seal and incubate at 46 1 C for 60 minutes To hybridize the Label Probe Step Action 1 Prepare Label Probe Working Reagent A Centrifuge Label Probe briefly to collect the contents to the bottom of the tube B Add 11 uL of Label Probe to 11 mL of Amplifier Label Probe Diluent C Invert to mix D Keep at room temperature until use Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 2B 2D two more times using 300 uL well of Wash Buffer Remove all traces of Wash Buffer A Place inverted plate with a dry paper towel into the centrifuge and centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available IMPORTANT Do not exceed 240 x g for 1 minute B Proceed to the next step immediately Add 100 uL of Label Probe Working Reagent to each well of the Capture
40. ted to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips 5 Add sample to the Capture plate Using a new pipette tip for each transfer add 40 uL of sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix IMPORTANT Add 40 uL of Homogenizing Solution to 3 wells for the assay background controls Run background wells for each Probe Set used 6 Bind target miRNA A Place an adhesive Plate Seal squarely on the plate and seal tightly IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 46 1 C incubator to begin the overnight 16 20 hour hybridization IMPORTANT Temperature must be 46 1 C for miRNA assay Verify temperature using a QuantiGene Incubator Temperature Validation Kit To capture target miRNA from purified RNA preparations Step Action 1 Prepare reagents a Probe Set s and Blocking Reagent Thaw vortex briefly to mix then briefly centrifuge to collect contents at the bottom of the tubes Keep on ice until use a RNA sample s If previously f
41. tion of the background fold change fold change Exp cell lysate pL subtracted RLU 60 3 100 31 3 103 20 1 000 2 70 3 90 6 6 370 Guidelines for Assay Optimization and Assay Design Overview Here we provide information and guidelines for Optimizing sample lysis Optimizing sample input Assay controls Assay replicates Calculations of miRNA copy number relative and absolute Optimizing Lysis Conditions To determine optimal sample amount for lysis or homogenization Step Action 1 Follow the recommended amount of cell number or tissue amount per volume of lysis mixture solution or homogenization solution listed in the Sample Processing Kit package insert for the specific sample types Recommendations are summarized below To ensure optimal lysis in the initial experiment run a test range as indicated in the table Recommended Sample Preparation Amount Cultured Cells Tissue Recommended 400 cells uL of Working 5 mg 300 uL of Working Lysis Mixture Tissue Homogenization Solution Test Range 200 400 800 cells uL of 2 5 5 0 10 mg 300 uL of Working Lysis Mixture Working Tissue Homogenization Solution 2 For each lysate prepare a 3 fold serial dilution to determine the assay performance Assay performance is determined by calculating the following u LOD u LOQ u Assay linearity assay CV 3 Calculate the assay performance for each sample to determine which one had
42. ured cell or whole blood PAXgene blood lysate s If previously frozen thaw at room temperature followed by incubation at 37 C for 15 30 minutes Vortex briefly then leave at room temperature until use a Lysis Mixture Re dissolve any precipitates by incubating at 37 C for 30 minutes followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 18 QuantiGene 2 0 miRNA Assay User Manual To capture target miRNA from cultured cell or blood lysates Step Action 2 Determine sample input Estimate sample input based on this chart and the LOD of miRNA target specific probe set from the package insert Recommended Sample Input based on LOD 3 000 Copies of miRNA Probe Set miRNA copies per cell Cultured Cells Whole Blood PAXgene number of cells Blood Lysate pL lt 10 6 000 80 100 600 80 gt 1 000 60 8 May not have sensitivity required If appropriate based on the expression level of target miRNA of interest dilute sample with Diluted Lysis Mixture 1 volume of Lysis Mixture plus 2 volumes of nuclease free water prepared fresh so that the final desired amount of sample present in a volume of 80 ul assay well Prepare Working Probe Set Prepare an appropriate volume of Working Probe Set by combining the foll
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