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Dual-Labeled Expression Microarray Protocol

1. Wash Buffer III Incubate for 15 seconds at RT with vigorous constant agitation Remove the slide from Wash III Load it active side up into the ArrayIT High Speed Microarray Centrifuge Centrifuge the slide for 1 minute Remove the slide from the carriage and return the dry hybridized slide to its cassette 9 Wash Buffer Il RT 1 min 10 Transfer slide to Wash Buffer III 11 Wash Buffer III RT 15 sec 12 13 14 Proceed immediately to scanning 33 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Two color Array Scanning Introduction Array scanning technique allows for imaging of fluorescently labeled DNA to produce biologically relevant data from microarray hybridizations Lasers excite the fluorescent dyes while photomultipler tubes record the illuminated pixels as a high resolution image file The protocol described here was adapted from Roche NimbleGen Array User s Guide for CGH and CNV Arrays The microarray is scanned using the NimbleGen MS 200 Microarray Scanner with resolution down to 2um Photomultipler tube PMT Gain is automatically adjusted by the Data Collection software to achieve consistent reproducible experimental results After image acquisition single tiff images are ready to load into NimbleScan 2 6 Software to grid images for data extraction Precautions Laboratory safety It is assumed that users have a sound kno
2. 2 3 For the 12 x 135K Array load the first hybridization reaction into AO1 fill port but do not over fill the array Load the hybridization reaction until it enters the vent port channel Do not allow the sample to come to the surface of the HX12 mixer Remove residual hybridization reaction surrounding the fill port and vent port with a Kimwipe Place the mixer seals using forceps over the fill and vent port Apply even pressure to the mixer seals to ensure a proper seal is achieved Continue to load and seal one chamber at a time until all samples have been loaded Close the hybridization system s bay clamp Be sure all slides have been processed hybridization reactions loaded mixer seals applied and hybridization bay clamps closed Turn on the mixing panel of the Hybridization System with the mix mode set to Mode B 30 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 4 Confirm the Hybridization System recognizes the slide in each occupied bay indicator light becomes green when hybridization bay is in use 5 Hybridize for 16 20 hours at 42 C mix Mode B 31 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Wash Hybridized Slides Instrument Setup Water bath 48 C Shallow dish P1000 tip box lid 500ml capacity NimbleGen Array Processing Containers NimbleGen Disassembly Tool ArrayIT Hig
3. 2 4 Place the spin filter in a microcentrifuge and spin for 10 minutes at 1500 rpm 2 5 Aliquot 65ul filtered gel into 0 5ml Rnase free tubes provided Store unused aliquots at 4 C and use within one month of preparation 3 Prepare gel dye mix 3 3 Vortex RNA 6000 Nano dye concentrate for 10 seconds and centrifuge briefly 3 4 Add lul of RNA 6000 Nano dye concentrate to 6511 aliquot of filtered gel Vortex well Centrifuge for 10 minutes at room temperate at 14000 rpm 3 5 Use gel dye mix immediately Otherwise store at 4 C for up one day Equilibrate to room temperature for 30 minutes Centrifuge gel dye mix for 10 minutes at 14 000 rpm prior to use 4 Load gel dye mix 4 3 Take anew RNA Nano chip out of its sealed bag 4 4 Place the chip on the priming station 4 5 Pipette 9 0ul of the gel dye mix from the upper layer of the 0 5ml microcentrifuge tube and dispense into the bottom of the well marked with the dark encircled G of the RNA Nano chip 4 5 1 When pipetting the gel dye mix from the 0 5ml microcentrifuge tube make sure not to draw up particles from the bottom of the gel dye mix tube Insert the tip of the pipette to the bottom of the chip well when dispensing This prevents a large air bubble forming under the gel dye mix Placing the pipette at the edge of the well may lead to poor results 49 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 4 6
4. THE CENTER FOR GENOMICS AND BIOINFORMATICS Dual Labeled Expression Microarray Protocol for High Throughput Genomic Investigations Jacqueline A Lopez amp John K Colbourne CGB Technical Report 2010 01 doi 10 2506 cgbtr 201001 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Introduction The utilization of microarrays for differential gene expression analysis focuses on protein encoding RNA of Prokaryote and Eukaryote systems Currently our workflow measures gene expression from test and reference samples of Eukaryote systems by dual color microarray hybridization The workflow is designed to detect steady state gene expression levels from limited samples Briefly RNA is obtained from the biological source of interest and processed to stable labeled targets for microarray hybridization from which a high resolution image is recorded and signal intensities are extracted and documented for analysis First Total RNA is extracted from the biological source with the use of organic solvents and purified with a silica based centrifugation column coupled with on column Dnase I treatment Next T7 based RNA amplification technique allows for linear amplification of polyA RNA from the limited sample Then double stranded cDNA amplification technique converts the amplified RNA into a suitable intermediate for random primer labeling After that NimbleGen Systems Hybridization t
5. Briefly Total RNA is extracted from the biological source with the use of organic solvents The extracted Total RNA is purified with a silica based centrifugation column The addition of on column based Dnase I digestion facilitates the removal of genomic DNA contamination At the completion of the procedure use of a spectrophotometer and microfluidic capillary electrophoresis assesses the quality of the purified Total RNA Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Caution TRIzol Reagent a phenol based solution is toxic when in contact with skin or if swallowed After contact with skin wash the area immediately with plenty of detergent and water This solution should be handled with caution Prevent excessive Rnase exposure As with all steps involved in RNA isolation it is essential to avoid latex glove Use only powder free nitrile gloves It is likewise important to guard against sources of dust and nucleases We recommend using Dnase and Rnase free plastics including barrier pipette tips In addition
6. Set the plunger is positioned at 1 ml and then close the chip priming station Depress the plunger to luer clamp Hold in place for 30 seconds Release the plunger clip Wait 5 seconds Return the plunger to 1ml position Open the Priming Station Load RNA 6000 Nano Marker 5 3 Pipette 5ul of the RNA 6000 Nano marker into the well marked with the ladder symbol and each of the sample wells to be used For unused sample wells pipette 6ul of the RNA 6000 Nano marker 5 3 1 Do not leave any wells empty or the chip will not run properly Unused wells must be filled with 6ul of the RNA 6000 Nano marker Load Ladder and Samples 6 3 Pipette lul of the RNA ladder into the well marked with the ladder symbol 6 4 Pipette lul of each sample into a separate sample wells Vortex the chip 7 3 Place the chip horizontally in the adapter of the IKA vortex mixer and secure the chip to the carriage Vortex the chip for 1 minutes at 2400 rpm Remove the chip from the vortex and remove any spilled liquid with a Kimwipe Insert Chip in the Agilent 2100 Bioanalyzer 8 3 Open the lid to the Agilent 2100 Bioanalzyer 8 4 Place the chip into the station 8 5 Gently close the lid to the Agilent 2100 Bioanalzyer Instrument Operation 1 Clicking START to begin the Chip Run 1 1 Ensure the appropriate assay has been selected 1 2 Adjust the wells to be measured setting if necessary 1 3 The runtime is approximately 25 minutes 7 minu
7. 05223547001 20 rxn DNA labeling B Mercaptoethanol Sigma Aldrich M3148 25mL 25 ml DNA labeling lsopropanol Sigma Aldrich 19516 500mL 500 ml Hybridization NimbleGen Hybridization Kit LS Roche NimbleGen 05583934001 40 slides Hybridization Mica ies cag nele Tackine Roche NimbleGen 05223512001 120 slides Control Kit Hybridization NimbleGen Wash Buffer Kit Roche NimbleGen 05584507001 10 uses Hybridization Mim BEGEN Aray Processing Roche NimbleGen 05223539001 lunlimited Accessories CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Hybridization Microman Capillany Pistonse CHLO laison F148412 192 tips nonsterile Hybridization ore ee A Peons ee Oe ien F148412 192 tips nonsterile Kvbridizations 1 are REaREnE RTAS AGS VWR RC91505 20L nonsterile Type Hybridization Kimwipe Cole Parmer EW 33670 04 Be eee boxes Hybridization Forceps Multiple vendors QA Agilent RNA 6000 Nano Kit Agilent 5067 1511 25 chips Supplies Rnase Zap Ambion AM9780 250 mL UltraPure Dnase Rnase Free N Supplies Distilled water Invitrogen 10977 015 500 mL Supplies PCR strip w cap 8 tubes strip VWR 20170 004 125 strips Supplies Premium Resedren MicrocEntrtugehi neci AVSS1700 1 000 Units tubes 1 5 ml Molecular Supplies ART 10 BioProducts 2139 960 Tips Molecular Supplies ART 20P BioProducts 2149P 960 Tips Mol
8. Agilent 2100 Bioanalyzer 9 4 Open the lid and place the electrode cleanser with nuclease free water into the Agilent 2100 Bioanalyzer Close the lid and leave it for 10 seconds Open the lid and remove the electrode cleanser chip from the Agilent 2100 Bioanalzyer Keep the lid open for 10 seconds then close the lid Setup Chip Priming Station 10 1 Replace the syringe 10 1 1 Replace the syringe with each new kit or after 25 chips have been processed 10 1 1 1 Unscrew the old syringe from the lid of the chip priming station 10 1 1 2 Release the old syringe from the clip Discard the old syringe 10 1 1 3 Remove the plastic cap of the new syringe and insert it into the clip 10 1 1 4 Slide it into the hole of the luer lock adapter and screw it tightly to the chip priming station 10 2 Adjust the base plate 10 2 1 Open the chip priming station by pulling the latch 10 2 2 Lift the base plate and insert it again in position C 10 3 Adjust the syringe clip 10 3 1 Release the lever of the clip and slide it up to the top position Proceed with Sample Preparation Sample Preparation Setup 1 Prepare Nucleic Acid sample s to a final concentration of 100ng ul 400ng ul 1 1 Total RNA 1 1 1 150ng per 1 5ul of nuclease free water in a 0 2mL micro centrifuge tube Mix by gently pipetting Centrifuge briefly to collect the contents 1 2 Amplified RNA aRNA 1 2 1 400ng per 1 5ul of nuclease free water in a 0 2mL micro
9. Control Image Control Roche button Application menu Figure 3 Top of Data Collection Workspace showing Scan Control Laser Control and Image Control Review the Magazine Control Figure 4 in the Data Collection Software Ensure that a green box appears in the Slide Present field for each slide loaded into the slide magazine 35 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 gt Sweremnae wa tor singe ov 2 opele 1 orty or a slides n p Magazine Control E Dula NerrBieGen ME SO Iregee oi Daima UserText LL A Piber VL gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt be UM PER SEE REE REERESSES ESE REESE REESE ESER ESE SES SE SSE ERS SI RNS VPOIOOOOGOGOG GOGO GOGGOGGGGGOV0OGHGGGGOGGG00G0000000000 10 2 io 10 10 1a 15 10 1a 2 1a 190 190 2 19 1a 10 10 15 19 1o 1 1 1 1 1 1 1 1 1 1 1 1 1 1a 10 1a 10 io 19 n 2 n bad n bad z 5 EJ a a a s a a Parameter File Settings Control Control Figure 4 Magazine Control Parameter Control and File Setting Control 8 Use the Parameter Control Figure 4 to se
10. D Random Primer Buffer with B 462 0uI Mercaptoethanol Total Volume 462 0ul 3 Aliquot 16 8ul individual reaction volumes in 0 2 ml thin walled PCR tubes Store at 20 C protect from light Yields approximately 25 individual reactions for each dye STOP POINT Diluted Cy Ramdon Nonamer may be stored for up to 4 months at 20 C until proceeding to next step Avoid freeze thaw cycles Assemble labeling reaction Instrument Setup 1 Program MasterCycler Thermocycler 1 1 98 C for 10 minutes lid temperature 105 C 1 2 37 C for 60 minutes x 2 lid temperature 105 C Sample Preparation Setup 1 Thaw the following reagents from Roche NimbleGen s Dual Color Labeling Kit unless otherwise noted 1 1 ON ICE Diluted Cy Random Nonamers 10mM dNTP mix Nuclease free water 1 2 Room temperature Stop Solution and 5M NaCl solution heat and vortex if precipitate is visible 2 Prepare 2 1 0 2ml thin walled PCR tube for each sample 23 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 2 2 Labeled 1 x 1 5ml microcentrifuge tube for Klenow Master Mix 2 3 Labeled 1 x 1 5ml microcentrifuge tube per sample 2 4 Labeled 1 x 1 5ml centrifuge tube per hybridization 2 5 Ethanol 80 Soln 3 All centrifugation steps are at room temperature unless otherwise noted Procedure 15 Prepare the test and reference samples in separate 0 2ml thin walled PCR tubes Dye as
11. and you 40 16 17 18 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 can remove the slide magazine Once the scan of a slide is completed a blue Displays Images button appears in the Display Images field of the Magazine Control Figure 4 Move the mouse pointer over the button to display the directory location and file name of the acquired image Click the blue Displays Images button to display the image both images in the Image View To view a small region of the array and to select where to view the histogram Figure 9 reduce the size of the magenta box that outlines the slide image in the Slide View so that the magenta box surrounds the area of interest The area of interest will appear in the Image View Click Show Hide Histogram to display the histogram Magenta Show Hide box Histogram button Histogram window Figure 9 Example Window Showing Magenta Box in the Side View Show Hide Histogram Button and Histogram Window Adjust contrast and brightness using the Image Control Figure 10 to provide an improved view of features on the image Adjusting these settings allows faint features to be more easily seen Show Lineprofile Vertical Show Lineprofile Horizontal Overlay Gamma 41 _ Brightness 0 H Contrast 100 D Channel 1 _ Channel 2 Figure 10 Image Control 4 CGB Technical Report 2010 01 Microarray Pro
12. centrifuge tube Mix by gently pipetting Centrifuge briefly to collect the contents 1 3 Double stranded cDNA 1 3 1 100ng per 1 5ul of nuclease free water in a 0 2mL micro centrifuge tube Mix by gently pipetting Centrifuge briefly to collect the contents 48 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 2 Prepare Nucleic Acid standard 2 1 RNA 6000 Ladder Agilent 2 1 1 Transfer 1 3ul to a 0 2mL micro centrifuge tube Mix by gently pipetting Centrifuge briefly to collect contents 3 Heat denature Nucleic Acid sample s and Nucleic Acid standard 3 1 70 C for 2 minutes 3 2 Immediately chill in ice bath 4 Keep Nucleic Acid sample s and Nucleic Acid standard on ice until ready to analyze 4 1 Analyze within 30 minutes Otherwise return samples to the freezer 20 C until ready to process Procedure 1 Allow all reagents from Bioanalyzer kit to equilibrate to room temperature protected from light for 30 minutes before use 1 1 RNA 6000 Nano Marker Green 1 2 RNA 6000 Nano dye concentrate Blue 1 3 RNA 6000 Gel Matrix 1 3 1 If filtered Gel Matrix is available equilibrate to room temperature for 30 minutes and skip prepare gel matrix 1 3 2 RNA 6000 Nano Gel Matrix Red 2 Prepare gel matrix Skip if filtered gel matrix is available 2 3 Place 550ul of Agilent 6000 Nano gel matrix into the top receptacle of a spin filter provided
13. dCTP dGTP and dTTP and bring to a final volume of 10001 with Nuclease free water 1 2 Store at 20 C for up to 6 months Sample Preparation Setup 1 Thaw the following reagents from Invitrogen unless otherwise noted 1 1 ON ICE Random Hexamer Primer Promega 0 1M DTT Soln 10mM dNTP mix 1 2 ROOM TEMP 5X First Strand Buffer 2 Prepare 2 1 1x 1 5ml microcentrifuge tube labeled First Strand Master Mix 2 2 0 2m thin walled PCR tube for each sample 3 All centrifugation steps are at room temperature unless otherwise noted 17 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 4 Remove the aRNA samples from 80 C storage Keep on dry ice until ready to proceed with double stranded cDNA synthesis Procedure 1 Prepare aRNA primer mixture in 0 2mL PCR tube Component Amount aRNA 10 0ug Random Primer 1 0ul Nuclease Free water To final volume Total Volume 11 0ul 2 Incubate aRNA primer mixture for 10 minutes at 70 C Immediately remove the RNA primer mixture from the thermocycler centrifuge briefly and chill for 5 minutes in an ice bath MasterCycler 70 C 10min 3 At room temperature prepare first strand master mix in a 1 5ml microcentrifuge tube labeled First Strand Master Mix Component Amount Blus 0 05 for error 5X First Strand Buffer 4 20ul 0 1M DTT Solution 2 10ul dNTP M
14. laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Caution Hybridization Component A containing formamide is toxic in contact with skin and if swallowed After contact with skin wash the area immediately with copious amounts of water This solution should be handled with caution Prevent excessive light exposure Cy labeled cDNA is light sensitive and should not be exposed to any unnecessary light When working with this component turn off all lights and close the blinds to the windows Prevent excessive ozone and humidity exposure Minimize ozone exposure and increased humidity levels as this leads to oxidation of the cyanine dyes Use of desiccant materials or a dehumidifier should be considered Instrument Setup 1 Heat block 95 C 2 Heat block 42 C 3 NimbleGen Hybridization System 42 C Sample Preparation Setup 1 Thaw on ice from the NimbleGen Hybridization Kit 1 1 2X Hybridization Buffer 1 2 Hybridization Component A 1 3 Alignment Oligo Soln 2 Thaw on ice from the NimbleGen Sample Tracking Control STC Kit 2 1 STC 1 3 5 7 9 11 13 15 16 17 18 20 for multiplex arrays only 3 Prepare 3 1 La
15. ml 30 ml Temperature 42 C RT RT RT 4 Pour Wash Buffer I 48 C into Shallow dish P1000 tip box lid 500ml capacity with Disassembly Tool Measure the temperature and wait until it reaches 42 C then proceed immediately with slide washing Heat Wash Buffer I if temperature is below 42 C Microwave may be used to adjust wash buffer temperature 32 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 5 Remove the Mixer Slide assembly from the Hybridization System and immediately load it into the Disassembly Tool Submerge the loaded Disassembly Tool into the Shallow dish containing warm Wash I 42 C Do not allow the slide to cool With the Mixer Slide assembly submerged carefully peel the mixer off the slide Discard the mixer While submerged in Wash Buffer I carefully remove the slide from the Disassembly Tool Gently agitate the slide for 10 seconds and quickly transfer the slide to Wash I RT 6 2 Itis important to agitate the slide in the warm Wash Buffer I 42 C This will facilitate the removal of hybridization reaction from the surface of the slide Failure to do so leads to poor washing results Wash Buffer I Incubate for 2 minutes at RT with vigorous constant agitation Wash Buffer RT 2 min 8 Transfer slide to Wash Buffer II Wash Buffer II Incubate for 1 minute at RT with vigorous constant agitation
16. session Save the Full Report 6 1 3 Browse for the file destination and enter an appropriate file name Click OK 45 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 7 Trouble shooting Fale T2 7 3 1 4 7 5 7 6 Make sure sample pedestal is clean Use 2 0ul of deionized water to wash pedestal and wipe dry with a KimWipe Redo the calibratation with nuclease free water step presented at the start of the software setup Redo the blank setup If blank measurement is not done properly strange results will occur Nucleic acid sample may not be homogenized Gently mixing the nucleic acid sample by finger flicking the micro centrifuge tube prior to measuring the concentration is recommended Re measure the sample Use a 1 5 2 0ul sample size when measuring Strange results occur when the liquid sample column is not completely formed during the measurement While making a measurement visually confirm the water column is completely formed If this does not resolve the issue the nucleic acid sample may need to be re purified Recommend Microcon YM 30 Centrifugal Filter Unit See Manufacturer s Manual 46 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Bioanalyzer RNA Protocol for NimbleGen Microarray Introduction Agilent Bioanalyzer system and reagent kits are designed to analys
17. to ensure complete dissociation of nucleoprotein complexes Repeat step 1 for the remaining frozen tissue samples to be processed Continue to step 2 after all tissue samples have been homogenized Add 200ul of Chloroform to each sample Vigorously shake the mixture for 15 seconds Do not vortex 3 Centrifuge the mixture for 15 minutes at 4 C at 11 600 rcf lt 12 000 x g Transfer upper aqueous phase approx 400u1 to labeled Rnase free 1 5ml Rnase free microcentrifuge tube supplied by user 5 Precipitate RNA by adding 0 5 volume approx 2001 of Absolute Ethanol 100 Soln Gently pipette 4 times to mix and flick tube 3 times Transfer precipitated RNA from the previous step to the labeled RNeasy mini spin column Proceed immediately Degradation and acidification of RNA can occur with prolonged expose at this point Maximum loading volume 700ul Maximum binding capacity 100ug 10 11 12 13 14 Centrifuge the column for 30 seconds at 10 000 rcf Discard flow through Replace column into same 2mL collection tube On Column Dnase Treatment Wash the column with 350ul of Buffer RW1 Centrifuge the column for 30 seconds at 10 000 rcf Discard flow through Replace column into same 2mL collection tube Pipette the Dnase I RDD Buffer mixture 40ul directly onto the RNeasy silica gel membrane of the RNeasy column Incubate for 10 minutes at room temperature Wash the column with another 350ul o
18. 12 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 MasterCycler 42 C 2hr 7 Remove the reaction from the thermocycler Centrifuge briefly Place reaction on ice Immediately proceed to second strand cDNA synthesis Second Strand cDNA Synthesis Instrument Setup 1 Program MasterCycler Thermocycler 1 1 16 C for 60 minutes x 2 heated lid off Sample Preparation Setup 1 Thaw the following reagents from MessageAmp II aRNA Amplification Kit part two 1 1 ON ICE Nuclease free water 10X Second Strand Buffer dNTP mix 2 Prepare 2 1 Labeled 1x 1 5ml microcentrifuge tube for Second Strand Master Mix 3 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Prepare second strand master mix on ice in a 1 5ml microcentrifuge tube labeled Second Strand Master Mix Component Brovided in kit Amount Plus 0 05 for part 2 error Nuclease Free water 66 15ul 10X Second Strand Buffer 10 50ul dNTP Mix 4 20u DNA Polymerase 2 10ul RNase H 1 05ul Total 84 0ul 2 Gently pipette the Second Strand Master Mix 4 times and flick 3 times to mix Centrifuge briefly Keep on ice 3 Add 80ul second strand master mix to the reaction 1001 total volume Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly 4 Incubate for 2 hours at 16 C heated lid off MasterCyc
19. 13 14 distributing the bead pellet Remove tube from the MagnaRack Add 460u1 Wash Buffer W12 Aspirate to resuspend the bead pellet without forming bubbles Place the sample on the MagnaRack for 1 minute until the beads form a pellet Without removing the tube from the MagnaRack carefully aspirate the supernatant without distributing the bead pellet Repeat Wash Procedure once more Remove tube from the MagnaRack Add 460ul Wash Buffer W12 Aspirate to resuspend the bead pellet without forming bubbles Place the sample on the MagnaRack for minute until the beads form a pellet Without removing the tube from the MagnaRack carefully aspirate the supernatant without distributing the bead pellet Remove tube from the MagnaRack Add 20ul Elution Buffer E5 Aspirate to resuspend the bead pellet without forming bubbles Incubate at room temperature for 1 minute Place the sample on the MagnaRack for 1 minute until the beads form a pellet Without removing the tube from the MagnaRack carefully transfer the supernatant containing the purified DNA to the second 1 5ml microcentrifuge tube without distributing the bead pellet STOP POINT Samples may be stored at 20 C until quality assessment 15 16 Determine concentration with NanoDrop See section NanoDrop Protocol for NimbleGen Microarray Determine integrity using BioAnalyzer RNA 6000 Nano kit See section Bioanalyzer RNA Protocol for NimbleGen Microarray STO
20. 5K Array Test Sample 6 0 ug 4 0 ug 4 0 ug Reference Sample 6 0 ug 4 0 ug 4 0 ug 32 SpeedVac on low heat protected from light until all the Nuclease free water is removed and the pooled labeled cDNA for hybridization is reduced to a pellet 25 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 STOP POINT Labeled cDNA pellet may be stored at 20 C protected from light until proceeding to next step 26 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 NimbleGen Systems Hybridization Introduction NimbleGen Systems Hybridization technique allows for uniform hybridization of labeled DNA to produce high interarray correlations between replicate hybridizations The protocol described here was adapted from Roche NimbleGen User s Guide for Expression Analysis for Cy labeled cDNA derived from Eukaryote systems We describe this procedure in detail for dual color competitive hybridization of single and multiplex array formats Briefly Cy labeled cDNA is hybridized to high density long oligonucleotide microarray using custom microarray slide mixer and specialized hybridization system At the completion of the procedure the hybridized microatray is ready for data collection Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe
21. Array 385K Array 2X Hybridization Buffer 29 5ul 88 5ul 11 8ul Hybridization Component A 11 8ul 35 4ul 4 7 ul Alignment Oligo 1 2ul 3 6ul 0 5ul Total 42 5ul 127 5ul 17 0pl 2 Add the appropriate amount of hybridization master mix to each rehydrated labeled cDNA according to the following table Component 4x72K Array 12x135K Array 385K Array Hybridization Master Mix 8 7 ul 8 7ul 13 0ul Total Volume 12 0pl 12 0pl 18 0pl Vortex well for 15 seconds and centrifuge briefly Incubate the hybridization mixture for 5 minutes at 95 C protected from light Incubate the hybridization mixture for 5 minutes at 42 C protected from light and maintain at 42 C until ready for sample loading Vortex well for 15 seconds and centrifuge prior to sample loading Prepare Mixer Slide assembly for hybridization Instrument Setup 1 2 NimbleGen Precision Mixer Alignment Tool PMAT NimbleGen Mixer Brayer 28 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 3 Forceps 4 NimbleGen Hybridization System 42 C Sample Preparation Setup 1 NimbleGen Array Mixer 2 NimbleChip Microarray Slide Procedure 1 Remove the appropriate mixer from its package Handle the mixer by the Tab with the NimbleGen barcode Avoid touching the body of the mixer because this will transfer unwanted fingerprints and other residues which may compromise array h
22. December 16 2010 6 Create Pair Reports Select ANALYZE gt PAIR Click ADD IMAGES to choose the files for analysis Select the design file ndf Browse to select the desired output file destination in the Choose the destination folder field Click REPORT NimbleScan creates one PAIR report pair for each image file 6 1 For 385K and 4x72K array formats select local alignment 6 2 For 12x135K array formats deselect local alignment 7 The PAIR report is the raw data of the array hybridization Other reports may be created For more information see NimbleScan Software User s Guide on http www nimblegen com gt Products gt Software gt literature 43 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 NanoDrop Protocol for NimbleGen Microarray Introduction The NanoDrop ND 1000 utilizes UV Vis spectroscopy to measures nucleic acid concentration The ND 1000 is a micro scale spectrophotometer that measures RNA or DNA concentration without dilution With a pre loaded application Nucleic Acid absorbance is measured across a broad wavelength range 190 840nm Values for 260nm absorbance determines nucleic acid concentration while 230nm and 280nm absorbance are used to calculate purity Precautions Prevent loss of data After report is generated it is recommended to save the report and copy the files to a secondary independent data storage device or location I
23. P POINT Samples may be stored at 20 C until proceeding to next step 21 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Random Primer Labeling Introduction Random Primer Labeling technique allows for amplification of ds DNA to produce enough material for microarray hybridization Procedures have been adapted to be compatible with genomic DNA and ds cDNA The protocol described here was adapted from Roche NimbleGen s Dual Color DNA Labeling Kit for amplification of ds cDNA derived from amplified RNA of Eukaryote systems We describe this procedure in detail along with DNA quality assessment measures Briefly ds cDNA is primed with CY labeled random nonamer and the addition of Klenow exo enzyme synthesizes 5 Cy labeled cDNA At the completion of the procedure use of a spectrophotometer assesses the quality of the purified 5 Cy labeled cDNA Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Caution Random Primer Buffer containing beta mer
24. Switch PCR Clean Up Kit 3 MagnaRack Magnetic Rack 4 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Add lul of 4mg ml RNase A Solution to the reaction Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly 1 1 Important Use caution when working with RNase A Use RNase ZAP to clean work area surfaces if necessary Component Amount Rnase A 4mg ml 1 0ul Total 163 5ul 2 Incubate for 10 minutes at 37 C lid temperature 105 C MasterCycler 37 C 10min 3 During the 10 minute incubation 3 1 add 163ul ChargeSwitch Purification Buffer N5 to the first labeled 1 5ml microcentrifuge tube and then set aside Component Amount Purification Buffer N5 163 0ul 4 Remove the reaction from the thermocycler Centrifuge briefly Transfer the reaction to the tube containing 163ul ChargeSwitch Purification Buffer N5 Aspirate to mix 20 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 5 Add 20ul ChargeSwitch Magnetic Beads to the Purification Buffer N5 reaction mixture Aspirate to mix without forming bubbles Incubate at room temperature for 1 minute Place the sample on the MagnaRack for 1 minute until the beads form a pellet 7 Without removing the tube from the MagnaRack carefully aspirate the supernatant without 10 11 12
25. Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 The latch s lever is in the up position when the latch is unlocked to allow for slide insertion _ While pressing the flap s flange insert each slide with the microarray side up and barcode end first into the slide magazine Ensure the barcode and NimbleGen logo are readable from the top Figure 1 Inserting Slides into the Slide Magazine Press the insert eject magazine button on the scanner to open the stacker cover Insert the slide magazine with loaded slides aligning the slot on the slide magazine s side to join with the rail profile inside the scanner The lowering of the slide magazine is interrupted by a mechanical hold point Apply gentle pressure to complete insertion Press the insert eject magazine button to close the stacker cover Figure 2 The initialization process starts checking the slide magazine to determine which slots are occupied Slot on the side of Slid zi the slide magazine ide magazine Insert eject magazine button Figure 2 Inserting the Slide Magazine into the Scanner Double click the NimbleGen MS200 icon to launch the MS 200 Data Collection Software Make sure that the software has completely loaded before continuing Click the Green Laser and Red Laser buttons in the Laser Control Figure 3 to switch on the lasers Allow lasers to warm for 10 minutes Help Scan Control Laser
26. Transcription Master Mix 2 2 0 2ml thin walled PCR tube for each sample 3 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Transfer the 16ul ds cDNA with T7 promoter site from previous section to 0 2ml PCR tube Centrifuge briefly Place on ice 2 Assemble the in vitro transcription master mix in 1 5ml microcentrifuge tube labeled In Vitro Transcription Master Mix at room temp in the order shown Componentprovided in kit Amountplus 0 05 part 2 for error T7 ATP Soln 75mM 4 2ul T7 CTP Soln 75mM 4 2ul T7 GTP Soln 75mM 4 2ul T7 UTP Soln 75mM 4 2ul T7 10X Reaction Buffer 4 2ul T7 Enzyme Mix 4 2ul Total 25 2ul 3 Transfer ds cDNA with T7 promoter site to room temperature Add 24ul of in vitro transcription master mix to each reaction 40ul total volume Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly 4 Incubate for 6 hours at 37 C followed by 4 C hold overnight MasterCycler 37 C 6hr 4 C HOLD 5 Remove from thermocycler Centrifuge briefly Add 60ul Nuclease free water to the reaction 1001 total volume Flick the reaction 3 times to mix Centrifuge briefly STOP POINT Reaction may be stored at 80 C overnight until purification however it is better to proceed to aRNA purification before stopping 15 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last up
27. Units RNA amplifica E EANET ARNA ANANA h AM1751 20 rxn Kit Part one amp two Double stranded CDNA Synthesis 100mM dNTP set 4x25umol Invitrogen 10297 018 1000 ul Double stranded CDNA Synthesis 5X Second Strand Buffer Invitrogen 10812 014 500 ul Double stranded chargeSwitch PCR Clean Up Kit CDNA Synthesis Invitrogen CS12000 100 rxn Double stranded 3 CDNA Synthesis DNA Polymerase 10U wl Invitrogen 18010 025 1000 Units Double stranded 7 l CDNA Synthesis E coli DNA Ligase 10U l Invitrogen 18052 019 100 Units Double stranded CDNA Synthesis Rnase H 2U ul 5U ul Invitrogen 18021 071 120 Units Double stranded i CDNA Synthesis RNASEOUT 40U nl Invitrogen 10777 019 5000 Units Double stranded CDNA Synthesis RT PCR Grade Water Invitrogen AM9935 1 5 ml x 10 Double stranded CDNA Synthesis SuperScript II 200U tl Invitrogen 18064 014 10000 Units Double stranded CDNA Synthesis T4 DNA Polymerase 5U tl Invitrogen 18005 025 250 Units Double stranded CDNA Synthesis Random Primer Promega C1181 20 ug Double stranded CDNA Synthesis Rnase A Solution 4 mg ml Promega A7973 1000 ul Double stranded i CDNA Synthesis 0 5M EDTA pH 8 0 Sigma Aldrich E7889 100mL 100 ml Double stranded 3 CDNA Synthesis 7 5M Ammonium Acetate Sigma Aldrich A2706 1L 1 liter POubIe strange ina Absola 200 proot Sigma Aldrich E7023 500mL 500 ml cDNA Synthesis y p g DNA labeling NimbleGen Dual Color Labeling Kit Roche NimbleGen
28. Z Andrea Yool William C Dement Jack D Barchas and James H Ederwine 1990 Amplified RNA synthesized from limited quantities of heterogeneous cDNA Biochemistry 87 1663 1667 N 1000 al cide S Acknowledgements This work was supported in part by the Indiana METACYt Initiative of Indiana University funded in part through a major grant from the Lilly Endowment Inc Portions of the microarray processing and analysis protocols provided in this document are courtesy of Roche NimbleGen Inc NimbleGen microarrays are intended for life science research only and are not for use in diagnostic procedures The protocol was developed by the authors of this document and it is not standard for processing NimbleGen Catalog Gene Expression microarrays We thank Tsetska Takova Director Global Marketing Arrays and Reagents at Roche NimbleGen Inc and Kary Staples Manager Global Marketing Communications at Roche NimbleGen Inc for help in creating this training manual and Jennifer Steinbachs CGB for help with formatting CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Total RNA Extraction and Purification Introduction The utilization of microarrays for gene expression analysis requires the extraction of RNA from a biological source of interest either from a Prokaryote or Eukaryote source We describe this procedure in detail along with RNA quality assessment measures
29. beled 1 x 1 5ml microcentrifuge tube for Hybridization Master Mix 4 All centrifugation steps are at room temperature unless otherwise noted 27 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Procedure 1 Set the Hybridization System to 42 C With the cover closed allow at least 3 hours for the temperature to stabilize Set one heat block to 95 C and a second heat block to 42 C allow at least 3 hours for the temperature to stabilize Rehydrate the dried labeled pooled cDNA pellet for hybridization 2 1 For multiple array platforms resuspend the dried labeled pooled cDNA pellet with the appropriate Sample Tracking Control STC Each hybridization reaction of the multiplex array should be resuspended with a unique STC Record the STC used for each hybridization reaction 2 2 For single array platforms resuspend the dried labeled pellet with Nuclease free water Component 4x72K Array 12x135K Array 385K Array STC Soln 3 3ul 3 3ul Nuclease free water 5 0ul Using components from the NimbleGen Hybridization Kit prepare the Hybridization master mix according to the following table The amounts listed in the table provide a sufficient volume 1 25x volumes to hybridize all arrays of a single slide When processing multiple slides adjust the amounts for the number and type of array Component 4x72K Array 12x135K
30. beling 1 98 C for 10 minutes lid temperature 105 C 2 37 C for 60 minutes x 2 lid temperature 105 C Quality Assessment QA 1 70 C for 2 minutes lid temperature 105 C CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 References 1 2003 Agilent 2100 Bioanalyzer 2100 Expert User s Guide Agilent Publication Number G2946 90004 2009 NimbleGen MS 200 Microarray Scanner Operator s Manual Roche NimbleGen http www nimblegen com lit 2006 Agilent RNA 6000 Nano Kit Guide Agilent Publication Number G2938 90034 2006 MessageAmp II aRNA Amplification Instruction Manual Ambion 2006 RNeasy Mini Handbook RNeasy Mini Kit QIAGEN 2007 ND 1000 Spectrophotometer V3 5 User s Manual NanoDrop Technologies 2010 NimbleScan Software User s Guide Roche NimbleGen http www nimblegen com lit 2007 TRIzol Reagent Invitrogen 2010 NimbleGen Arrays User s Guide CGH Analysis Roche NimbleGen http www nimblegen com lit 10 2008 SuperScript Double stranded cDNA Synthesis Kit Invitrogen 11 2010 NimbleGen Arrays User s Guide Expression Analysis http www nimblegen com lit 12 2010 NimbleGen Hybridization Systems User s Guide Roche NimbleGen http www nimblegen com lit 13 J Sambrock and D W Russell Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 14 Russell N Van Gelder M E V
31. captoethanol after preparation is toxic in contact with skin and if swallowed After contact with skin wash the area immediately with copious amounts of water This solution should be handled with caution Prevent excessive light exposure Cy labeled Random Primer reagent and subsequent Cy labeled cDNA is light sensitive and should not be exposed to any unnecessary light When working with the labeled components turn off all lights and close the blinds to the windows Prevent excessive ozone and humidity exposure Minimize ozone exposure and avoid humidity levels above 40 as this leads to oxidation of the cyanine dyes Use of desiccant materials or a dehumidifier should be considered 22 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Sample Preparation Setup 1 Thaw the following reagents from Roche NimbleGen Dual Color Labeling Kit unless otherwise noted 1 1 ON ICE Random Primer Buffer 2 Prepare 2 1 50 x 0 2ml thin walled PCR tube 3 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Prepare Random Primer Buffer Component Amount Random Primer Buffer 998 25ul B Mercaptoethanol 1 75ul Total Volume 1000 0ul 2 Centrifuge briefly Cy3 and Cy5 Random Nonamer Dilute each Cy primer with 462ul of Random Primer Buffer with B Mercaptoethanol Component Amount Cy Random Nonamer 11 O
32. dated December 16 2010 aRNA Purification Instrument Setup 1 Heat block 55 C Sample Preparation Setup 2 MessageAmplII aRNA Amplification Kit part one 2 1 aRNA Binding Buffer 2 2 Wash Buffer ethanol added as directed by manufacturer 2 3 Nuclease free water 2 4 Labeled 1 x 2ml aRNA filter cartridge with collection tube per sample 2 5 Labeled 1 x 2ml aRNA collection tube per sample 3 Prepare 3 1 Labeled 1 x 1 5ml microcentrifuge tube per reaction for precipitation 3 2 Absolute Ethanol 100 Soln 4 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Preheat Nuclease free water to 55 C 2 Assemble aRNA Filter Cartridges and aRNA collection tubes 3 Transfer the in vitro transcription reaction to labeled 1 5m1 microcentrifuge tube for precipitation 4 Add 350ul aRNA Binding Buffer to reaction 5 Add 250ul Absolute Ethanol 100 Soln to reaction Proceed immediately to the next step 6 Pipette the precipitation mixture 4 times to mix and transfer to aRNA Filter Cartridge Centrifuge for 1 minute at 10 000 rcf Discard the flow through Replace the aRNA Filter Cartridge into the same aRNA Collection tube 7 Wash column with 650ul Wash Buffer Centrifuge for 1 minute at 10 000 rcf Discard the flow through Replace the aRNA Filter Cartridge in the same aRNA Collection tube 8 Centrifuge for 1 minute at 10 000 rcf to remove trace amounts of wash buffer 9 Transfer aRNA F
33. decontaminate both workspaces and pipettes with RNase Zap according to manufacturer s instructions Instrument Setup 1 Chill a refrigerated centrifuge to 4 C 2 Equilibrate TRIzol Reagent to room temperature for at least 1 hour prior to use Sample Preparation Setup 1 For each sample 1 1 Labeled 1 x 1 5ml Rnase free microcentrifuge tube 1 2 Labeled 1 x 1 5ml RNeasy collection tube 1 3 Labeled 1 x RNeasy mini spin column plus an additional 2ml collection tube 1 4 Rnase free Dnase Kit Add 35ul of RDD Buffer to Sul of Dnase I stock solution Mix by gently pipetting Store at 4 C for up to one day Rnase free Dnase Kit Reconstitute lyophilized Dnase I as directed by manufacturer RNeasy Mini Kit Add Ethanol 100 Soln to Buffer RPE as directed by manufacturer All centrifugation steps are at room temperature unless otherwise noted Ne se Remove the frozen tissue samples from 80 C storage Keep on dry ice until ready to perform extraction procedure CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Procedure l Add 500u1 of TRIzol Reagent directly to the tube containing the frozen tissue sample Immediately homogenize tissue Grind tissue using disposable blue pestle After tissue has been completely homogenized add another 500ul of TRIzol Reagent to the tube containing the homogenized tissue Incubate mixture at least 5 minutes at room temperature
34. e at www nimblegen com scanner under Download These files identify scan areas that are specific for NimbleGen 385K or 4x72K arrays For instructions on how to use area files or how to manually adjust the scan area refer to the NimbleGen MS 200 Microarray Scanner Operator s Manual If desired click Save to save settings to an area file for future use d Review and if necessary adjust the autogain area 38 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 To adjust the autogain area position the mouse pointer inside the respective rectangle and click to enable the move cursor Drag the rectangle to the desired array location and about 10 mm from the edge of the default scan area Size the rectangle by dragging the side and corner handles 3 mm x 3 mm to 22 mm x 22 mm Click Apply to confirm your settings Figure 6 Example of Autogain Areas for NimbleGen Arrays 11 Repeat the instructions above for each slide to process during the experiment If you saved parameter file s and area file s click Load in the Parameter Control or Area Definition Control respectively to select a file and then click Apply to confirm your selection 12 Click the Start Stop Scan button in the Scan Control Figure 7 13 When prompted specify the folder and file naming to save the session file The scan process is then initiated The Parameter Control and Area Definition Control c
35. echnique allows for uniform hybridization of the labeled targets to produce high interarray correlations between replicate hybridizations Subsequently array scanning excites the fluorescent dyes to record a high resolution image file from which NimbleScan Software extracts quantifiable data for analysis At the completion of the procedure data is presented in a format suitable for normalization and comparative analysis Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Materials Application Item Name Company Catalog Number Unit Size RNA Isolation Chloroform EMD Chemicals CX1055 6 500 ml RNA Isolation TRIzol Reagent Invitrogen 15596 026 100 ml RNA Isolation Rnase free Dnase set 50 Qiagen 79254 50 rxn RNA Isolation RNeasy Mini Kit Qiagen 74104 50 rxn RNA Isolation Disposable pestles 1 5mL Plastic VWR KT749521 1590 1100
36. ecular Supplies ART 200 BioProducts 2069 960 Tips Molecular Supplies ART 1000E BioProducts 7029E 800 Tips Required for 4x72K and 12x135K array formats only Required for 385K array format only CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Equipment Capacity Application Item Name Company Catalog Number Version Sample Processing Mastercycler gradient Eppendorf 5331 000 010 0 2ml x 96 Sample Processing Microcentrifuge 5424 Eppendorf 22620401 1 5ml x 24 Sample Processing Microcentrifuge 5415 R Eppendorf 5425 725 000 1 5ml x 24 Sample Processing Vacuum Centrifuge Multiple vendors 1 5ml x 24 Sample Processing Microcentrifuge Multiple vendors ae Sample Processing Heat block Multiple vendors 1 5ml x 24 Sample Processing Water bath Multiple vendors 20L Sample Processing MagnaRack Magnetic Rack Invitrogen ICS15000 1 5ml x 12 Hybridization Microman M10 Pipette Gilson F148501 Hybridization Microman M100 Pipette Glison F148504 Hybridization Hybridization System 4 110V Roche NimbleGen 5223652001 4 slides Hybridization Precision Mixer Alignment Tool Roche NimbleGen Included with hyb 1 slide PMAT system Hybridization Disassembly tool Roche NimbleGen included witi hyb 1 slide systems Hybridization NimbleGen MS 200 Microartai ji che hlimbleGen 05394341001 Hg shdes Sca
37. ent Amount Stop Solution 4 0ul Total 44 0ul 24 Add 4 6ul of 5M NaCl to the reaction Vortex to mix and centrifuge briefly Component Amount 5M NaCl 4 6ul Total 48 6ul 25 Transfer the reaction to labeled 1 5ml microcentrifuge tube containing 44ul of Isopropanol for precipitation Vortex well Component Amount lsopropanol 44 0ul Total 92 6ul 26 Incubate for 10 minutes at room temperature protected from light Centrifuge for 10 minutes at 12 000 rcf Decant supernatant 27 Wash pellet with 500ul of 80 Ethanol Solution v v Centrifuge for 2 minutes at 12 000 rcf Decant supernatant 28 SpeedVac the pellet on low heat for 5 minutes to remove traces of wash buffer STOP POINT Labeled pellet may be stored at 20 C for up to 5 days protected from light until proceeding to next step 29 Centrifuge briefly before opening Rehydrate pellet with 12 5ul of Nuclease free water Incubate at room temperature protected from light for 5 minutes Gently vortex and centrifuge briefly 30 Determine concentration with NanoDrop See section NanoDrop Protocol for NimbleGen Microarray 31 Based on the concentration calculate the volume of the test sample and reference sample required per hybridization based on the following table Combine both test and reference samples into labeled 1 5ml microcentrifuge tube for hybridization Sample Requirement 385K Array 4x72K Array 12x13
38. f Buffer RW1 Centrifuge the column for 30 seconds at 10 000 rcf Discard flow through Replace column into same 2mL collection tube Wash the column with 500ul of Buffer RPE Centrifuge the column for 30 seconds at 10 000 rcf Discard flow through Replace column into same 2mL collection tube Wash the column with another 500ul of Buffer RPE Centrifuge the column for 30 seconds at 10 000 rcf Discard flow through Place column ina NEW 2mL collection tube Centrifuge for 2 minutes at 10 000 rcf Transfer the column to the labeled 1 5ml collection tube supplied in Rneasy kit To elute pipette 30ul of Rnase free water directly onto silica gel membrane of the column Incubate for 1 minute at room temperature Centrifuge the column for 1 minute at 10 000 rcf The elutate now contains purified Total RNA STOP POINT Samples may be stored at 80 C until quality assessment is completed CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 15 Determine concentration with NanoDrop See section NanoDrop Protocol for NimbleGen Microarray 16 Determine integrity using BioAnalyzer RNA 6000 Nano kit See section Bioanalyzer RNA Protocol for NimbleGen Microarray STOP POINT Samples may be stored at 80 C until ready for processing 10 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 MessageAmp II aRNA Amplificat
39. ffer 31 50ul dNTP Mix 10mM 3 15 E coli DNA Ligase 10U tl 1 05ul E coli DNA Polymerase 10U tl 4 20ul E coli Rnase H 2U ul 1 05ul Total 136 5ul 2 Add 130ul Second Strand Master Mix to reaction 150 5 total volume Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly 3 Incubate or 2 hours at 16 C heated lid off MasterCycler 16 C 2hr 4 At the pause in the cycle add 2ul E coli T4 DNA Polymerase to reaction 152 5 total volume Gently pipette 4 times to mix Component Amount E coli T4 DNA Polymerase 5U tl 2 0ul Total 152 5ul 5 Resume thermocycler program Incubate for 5 minutes at 16 C heated lid off MasterCycler 16 C Smin 19 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 6 Remove the reaction from the thermocycler Centrifuge briefly Add 10ul of 0 5M EDTA pH 8 0 Flick tube 3 times to mix Centrifuge briefly Component Amount 0 5M EDTA pH 8 0 10 0ul Total 162 5ul STOP POINT Samples may be stored at 20 C overnight however it is better to complete the cDNA purification before stopping Rnase A Treatment and Clean Up Instrument Setup 1 Program MasterCycler Thermocycler 1 1 37 C for 10 minutes lid temperature 105 C Sample Preparation Setup 1 Prepare 1 1 Labeled 2 x 1 5ml microcentrifuge tube per reaction 2 Charge
40. h ON the computer system operating the Bioanalyzer 2100 System and Bioanalyzer 2100 Expert Software 4 Launch Bioanalyzer 2100 Expert Software 5 After the software opens the Instrument control panel will appear 6 Make sure the Bioanalyzer 2100 System has been detected 6 1 From instrument control panel select the instrument 7 Click on METHODS select the method for measurement 7 1 Eukaryote Total RNA Nano 7 1 1 Evaluates 18S and 28S based Eukaryote RNA 7 2 Prokaryote Total RNA Nano 7 2 1 Evaluates 16S and 23S based Prokaryote RNA 7 3 mRNA Nano 7 3 1 Evaluates distribution of nucleic acid molecule to detect ribosomal RNA contamination for amplified RNA aRNA and double stranded ds cDNA 47 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 8 10 11 Input Chip Information 8 1 Sample Name 8 2 Sample Comments 8 3 Chip Lot Number 8 4 Kit Lot Number 8 5 Comments Wash the electrode pins and allow drying for 10 minutes 9 1 Obtain electrode cleansers from the Bioanalzyer Kit label one RnaseZap and label the other Nuclease free water 9 2 Slowly fill the first with RnaseZap 350ul and the second with nuclease free water 350ul 9 3 Open the lid and place the electrode cleanser with RnaseZap into the Agilent 2100 Bioanalyzer Close the lid and leave it for 1 minute Open the lid and remove the electrode cleanser chip from the
41. h Speed Microarray Centrifuge PE NS Sample Preparation Setup 1 1X NimbleGen Wash Buffer I 42 C amp RT 2 1X NimbleGen Wash Buffer II 3 1X NimbleGen Wash Buffer III 4 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 IMPORTANT for ALL Washing Steps 1 1 Wash one slide at a time since the ArrayIT High Speed Microarray Centrifuge accommodates only one slide at a time Restart washing protocol when each microarray slide is dry 1 2 Handle the slide from the barcode edge with a firm grip do not use forceps Barcode edge is at the far end of the Mixer Disassembly Tool so the slide will have to be turned around 180 degrees before inserting into wash containers When transferring the slides into the next wash buffer minimize the amount of wash buffer carryover by tapping the edge of the slide gently on a Kimwipe 2 Setup ArrayIT High Speed Microarray Centrifuge with slide carriage 3 Prepare NimbleGen Hybridization Wash Buffers 3 1 Pre warm overnight 270ml of 1X Wash Buffer I in a water bath set to 48 C 3 2 Prepare 1X NimbleGen Wash Buffers in NimbleGen Array Processing containers 3 2 1 For the centrifuge tube with blue cap final volume is 27ml SMALL SCALE 1 slide Component Wash Wash Wash II Wash III VWR water Type 270 ml 27 ml 27 ml 27 ml 10X NimbleGen Wash Buffer 30 ml 3 ml 3 ml 3 ml 1M DTT Soln 30 ul 3 ul 3 ul 3 ul Total 300 ml 30 ml 30
42. hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Caution Ambion s aRNA binding Buffer containing beta mercaptoethanol is toxic when in contact with skin or if swallowed After contact with skin wash the area immediately with copious amounts of water This solution should be handled with caution Prevent excessive Rnase exposure As per all steps involved in RNA amplification it is essential to avoid latex glove Use only powder free nitrile gloves It is likewise important to guard against sources of dust and nucleases We recommend using Dnase and Rnase free plastics including barrier pipette tips In addition decontaminate both workspaces and pipettes with RNase Zap according to manufacturer s instructions Instrument Setup Reverse Transcription to Synthesize First Strand CDNA Instrument Setup 1 Program MasterCycler Thermocycler 1 1 70 C for 10 minutes lid temperature 105 C 1 2 42 C for 60 minutes x 2 lid temperature 50 C 11 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Sample Preparation Setup 1 Thaw the following reagents from MessageAmp II aRNA Amplification Kit part two 1 1 ON ICE T7 Oligo dT Primer dNTP mix 1 2 ROOM TEMP 10X First Strand Buffer Prepare 2 1 Labeled 1 x 1 5ml mic
43. hrogram of aRNA 3 High Quality ds cDNA Profile 3 1 One marker peak 3 2 Broad distribution medial molecular weight molecules 3 3 Minimal low molecular weight molecules 3 4 Minimal ribosomal RNA contamination lt 5 25 200 1000 4000 nt Figure 3 Electophrogram of ds cDNA 52
44. id introducing bubbles into the array When ready slowly dispense the appropriate loading volume into the fill port until the hybridization reaction starts to leak out of the array s corresponding vent port Using a KimWipe carefully to remove residual hybridization reaction surrounding the fill port and vent port Adhered the mixer seal over the fill port and vent port Close the hybridization system s bay clamp Component 4x72K Array 12x135K Array 385K Array Loading Volume 8 0ul 6 0ul 16 0ul Pipette amp Tip CP10 CP10 CP100 2 1 For the 385K Array load the hybridization reaction and remove residual hybridization reaction surrounding the fill port and venting port with a Kimwipe Place the mixer seals using forceps over the fill port and vent port Apply simultaneous even pressure to the mixer seals to ensure a proper seal is achieved Close the hybridization system s bay clamp 2 2 For the 4 x 72K Array load the first hybridization reaction into AO1 fill port and remove residual hybridization reaction surrounding the fill port and vent port with a Kimwipe Repeat loading for hybridizations reactions into the A02 A04 fill ports using a fresh Kimwipe for each array After the last hybridization reaction is loaded place the mixer seals using forceps over the fill port and vent ports Apply even pressure to the mixer seals to ensure a proper seal is achieved Close the hybridization system s bay clamp
45. ilter Cartridges to labeled 2ml aRNA Collection tube for elution 10 Add 100ul Nuclease Free Water preheated to 55 C to the center of the filter of the aRNA Filter Cartridge 11 Incubate for 2 minutes at 55 C and then centrifuge for 1 5 minutes at 10 000 rcf 12 The elutate now contains purified aRNA STOP POINT Samples may be stored at 80 C until quality assessment 13 Determine concentration with NanoDrop See section NanoDrop Protocol for NimbleGen Microarray 14 Determine integrity using BioAnalyzer RNA 6000 Nano kit See section Bioanalyzer RNA Protocol for NimbleGen Microarray STOP POINT Samples may be stored at 80 C until proceeding to next step 16 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Double stranded cDNA Synthesis Introduction Double stranded cDNA Synthesis technique allows for amplification of RNA to produce enough material suitable for random primer labeling The protocol described here was adapted from Invitrogen s SuperScript Double Stranded cDNA Synthesis Kit for amplification of aRNA derived from Eukaryote systems We describe this procedure in detail along with RNA quality assessment measures Briefly aRNA is primed with random hexamer primer in a reverse transcription reaction to synthesize first strand cDNA Second strand cDNA synthesis completes the strand to produce double stranded cDNA In the presence of T4 DNA Polyme
46. ion Introduction The utilization of microarrays for transcription analysis requires large amounts of RNA from a biological source of interest T7 based RNA Amplification technique originally developed in the laboratory of James Eberwine Russell N Van Gelder 1990 allows for linear amplification of polyA RNA from limited samples to produce enough material for microarray hybridizations Procedures have been adapted to be compatible with Prokaryote or Eukaryote systems The protocol described here was adapted from Ambion s MessageAmp II Amplification kit for amplification of polyA RNA from Eukaryote systems We describe this procedure in detail along with RNA quality assessment measures Briefly Total RNA is reverse transcribed with a T7 Oligo dT primer to synthesis first strand cDNA T7 promoter site is completed by second strand cDNA synthesis The RNA Polymerase driven in vitro transcription converts the double stranded cDNA with T7 RNA Polymerase promoter site into the final product amplified antisense RNA aRNA At the completion of the procedure use of a spectrophotometer and microfluidic capillary electrophoresis assesses the quality of the purified aRNA Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential
47. is limited samples by microfluidic capillary electrophoresis By replacing ethidium bromide with fluorescent dye the system is a safer alternative for nucleic acid analysis Standardized analysis applications included with the analysis software provide reliable data which can be compared between independent chip runs Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Caution RNA 6000 dye concentrate contains DMSO a molecule that facilitates chemical absorption into cells All materials should be handled with caution Prevent loss of data After report is generated it is recommended to save the report and copy the files to a secondary independent data storage device or location Instrument Setup 1 Confirm the electrophoresis cartridge is installed in the Bioanalyzer 2100 System 1 1 Electrophoresis cartridge is 1 2 Switch ON the Bioanalyzer 2100 System 2 1 Green Light ON and ready for measurement 2 2 Green Blinking Measuring 2 3 Orange Blinking Busy not measuring 2 4 Red Light Not ready 3 Switc
48. ix 10mM 1 051 RNaseOUT 40U ul 0 525ul SuperScript II 200U tl 2 10ul Total Volume 9 975ul 4 Transfer aRNA primer mixture to room temperature Add 9 5ul First Strand Master Mix to aRNA primer mixture 20 51 total volume Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly 5 Incubate for 60 minutes at 42 C lid temperature 50 C MasterCycler 42 C 60min 6 Remove the reaction from the thermocycler Centrifuge briefly Place the reaction on ice Proceed immediately to second strand cDNA synthesis 18 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Second Strand cDNA Synthesis Instrument Setup 1 Program MasterCycler Thermocycler 1 1 16 C for 60 minutes x 2 heated lid off then Pause followed by 16 C for 5 minutes heated lid off and finally 4 C Hold Sample Preparation Setup 1 Thaw the following reagents from Invitrogen unless otherwise noted 1 1 ON ICE Nuclease free water 5X Second Strand Buffer dNTP mix 2 Prepare 2 1 Labeled 1 x 1 5ml microcentrifuge tube for Second Strand Master Mix 3 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Prepare second strand master mix on ice in a 1 5ml microcentrifuge tube labeled Second Strand Master Mix Component 0 0596 for error Nuclease Free water 95 55ul 5X Second Strand Bu
49. l click the Open Close File Settings button 8 to open the File Settings Control b Choose the Single TIFF option button to generate one image file in Tagged Image File Format TIFF tif per channel c Ifnecessary change the path to which the images files will be saved The default path is E Data NimbleGen MS 200 To change the path click Browse to open a dialog box to specify a location in the directory and click OK to confirm d Use the annotation list to add or change annotations to include in file names Click an annotation in the left list box and then click the right arrow button 8 to add to the right list box If you will be using NimbleScan software for data analysis specify and order the annotations as follows lt Barcode gt _ lt User Text gt _ lt Laser WL gt tif where WL means wavelength To change the order of the annotations select the annotation and click the Up or Down button For User Text make sure to type the text in the User Text field The Filename Preview text box of the Parameter Control displays the entire naming convention of the image file tif e Click Apply to confirm settings f Optional To save the file settings to a file click Save in the Parameters Control Figure 4 10 Use the Area Definition Control Figure 5 denoted as Scan Areas in the software interface to set scan barcode and autogain areas a Toopen the Area Definition Control if not displayed in the Parameter Co
50. ler 16 C 2hr 5 Remove the reaction from thermocycler Centrifuge briefly Place the reaction on ice STOP POINT Reaction may be stored at 20 C overnight however it is better to complete the cDNA purification before stopping 13 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 cDNA Purification Instrument Setup 1 Heat block 53 C Sample Preparation Setup 1 MessageAmplII aRNA Amplification Kit part one 1 1 cDNA Binding Buffer 1 2 Wash Buffer ethanol added as directed by manufacturer 1 3 Nuclease free water 1 4 Labeled 1 x 2ml cDNA filter cartridge with cDNA collection tube per sample 1 5 Labeled 1 x 2ml cDNA collection tube per sample 2 Ifa precipitate is visible in the cDNA Binding Buffer warm solution to 37 C for up to 10 minutes Cool to room temperature before use 3 Prepare 3 1 Labeled 1 x 1 5ml microcentrifuge tube per reaction for precipitation 4 All centrifugation steps are at room temperature unless otherwise noted Procedure 1 Preheat Nuclease free water to 53 C for at least 10 minutes 2 Assemble cDNA Filter Cartridges and 2ml cDNA collection tubes 3 Transfer cDNA reaction to labeled 1 5ml microcentrifuge tube for precipitation 4 If necessary adjust cDNA reaction volume to 100ul with Nuclease free water 5 Add 250ul of cDNA Binding buffer to cDNA reaction 6 Pipette the precipitation mixture 4 ti
51. lose The Image View Slide View and Spot View open Figure 7 and the Scan Control shows the current task that is being performed above the progress bar 39 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Sean Spot Image Control Control LULL OeUUSUOSOCUIPSESSSISTISIIISISTIEri rit iret iiiy TT T TTT TTT TTT TETT Slide View Image View Figure 7 Example of a Run Time Window 14 Optional To manually adjust the PMT gain while scanning at 5 um or higher resolution a Use the Gain 1 and Gain 2 spin boxes that appear in the Scan Control Figure 7 to adjust the gain pee Gain 2 100 100 Click Show Histogram in the Image Control Figure 7 to view the histogram Use the zoom feature magenta box in the Slide View Figure 9 to select where to view the histogram The best data are achieved when the red and green curves are superimposed or as close as possible to one another If the red curve is above the green curve lower the red curve Channel 2 The curves should level out near 3 logio Ph eee cre sise otoc ese woe ener mess omi Figure 8 Histogram Window c Record the final gain settings to use to scan the slide again 15 After the scanning experiment is completed the light in the upper left corner of the insert eject magazine button on the scanner becomes green The scanner unlocks its stacker cover
52. mes to mix and transfer to the cDNA Filter Cartridge Centrifuge for 1 minute at 10 000 rcf Discard the flow through Replace the cDNA Filter Cartridge in the same collection tube 7 Wash the column with 500ul Wash Buffer Centrifuge for 1 minute at 10 000 ref Discard the flow through Replace the cDNA Filter Cartridge in the same collection tube 8 Centrifuge for 1 minute at 10 000 rcf to remove trace amounts of wash buffer Transfer cDNA Filter Cartridge to labeled 2ml cDNA collection tube for elution 9 Add 18ul of Nuclease free Water preheated to 53 C to the center of the filter in the cDNA Filter Cartridge 10 Incubate for 2 min at 53 C Centrifuge for 1 5 minutes at 10 000 rcf 11 The elutate now contains purified double stranded cDNA with T7 promoter site STOP POINT Samples may be stored at 20 C overnight however it is better to proceed to In Vitro Transcription before stopping 14 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 In Vitro Transcription Instrument Setup 1 Program MasterCycler Thermocycler 1 1 37 C for 60 minutes x 6 lid temperature 105 C 4 C Hold Sample Preparation Setup 1 Thaw the following reagents from MessageAmp II aRNA Amplification Kit part two 1 1 ON ICE T7 ATP Soln T7 CTP Soln T7 GTP Soln T7 UTP Soln 1 2 ROOM TEMP 10X Reaction Buffer 2 Prepare 2 1 Labeled 1 x 1 5ml microcentrifuge tube for In Vitro
53. nants that absorb at or near 280 nm 5 1 2 260 230 ratio range of 1 8 2 2 5 1 2 1 If the ratio is low this may indicate the presence of co purified contaminants e g Mg2 salts or other wash buffers 5 1 3 Spectrophotometer graph shows smooth curve 44 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 5 2 Fords cDNA and Cy labeled cDNA 5 2 1 260 280 ratio of gt 2 0 5 2 1 1 If the ratio is lower this may indicate the presence of protein phenol or other contaminants that absorb at or near 280 nm 5 2 2 260 230 ratio range of 1 8 2 2 5 2 2 1 If the ratio is low this may indicate the presence of co purified contaminants e g Mg2 salts or other wash buffers 6 Save Report 6 1 From Menu Bar at top of window select REPORTS and click Save Report 6 1 1 Select Export Table Option 2 for a tab delimited text file 6 1 1 1 This file can be viewed in MS Excel application 6 1 2 Select Full Report for a NanoDrop formatted file 6 1 2 1 This file can be viewed in the NanoDrop software only In addition the report can be loaded at the start of the next session if a single continuous record file is desired 6 1 2 2 To load previous full report select REPORTS and click Load Report Browse for the desired report select it and click OK to load it Once loaded measurements taken during the new session will add to the report At the end of the
54. nner Hybridization Microarray High Speed Centrifuge ArrayIT MHC110V 1 Slide QA NanoDrop 1000 ThermoScientific SID 10135606 1 sample QA Bioanalyzer 2100 Agilent G2938C 1 chip QA Chip Priming Station Agilent 5065 4401 1 chip QA IKA Vortex Chip Adapter Agilent 5065 9966 1 chip soRiare NimbleGen MS 200 Data Collection Roche NimbleGen L 11 Software Software NimbleScan Roche NimbleGen 05 933 315 001 12 6 Software NanoDrop 1000 Software ThermoScientific 2 5 Software Bioanalyzer Software Agilent G2941AA B 02 05 Required for 4x72K and 12x135K array formats only Required for 385K array format only CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Workflow CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Thermocycler Programs MessageAmp II aRNA Amplification 1 70 C for 10 minutes lid temperature 105 C 2 42 C for 60 minutes x 2 lid temperature 50 C 3 16 C for 60 minutes x 2 heated lid off 4 37 C for 60 minutes x 6 lid temperature 105 C 4 C Hold Double stranded cDNA Synthesis 1 70 C for 10 minutes lid temperature 105 C 2 42 C for 60 minutes lid temperature 50 C 3 16 C for 60 minutes x 2 heated lid off then Pause followed by 16 C for 5 minutes heated lid off and finally 4 C Hold 4 37 C for 10 minutes lid temperature 105 C Random Primer La
55. nstrument Setup 1 Launch Nanodrop software to start application and adjust setting 2 1 Select assay Nucleic Acid 3 A prompt will appear Following the instructions from the prompt 3 1 With a KimWipe gently buff the top and bottom pedestal 5 times 3 2 Load 1 5ul of nuclease free water to pedestal Gently lower arm and click OK Procedure 1 Select Sample Type from drop down menu displayed at the top left of the measurement window 1 1 For Total RNA and aRNA quantification 1 1 1 Select Sample type RNA 40 for extinction coefficient 40 1 2 For DNA quantification i e ds cDNA and Cy labeled cDNA 1 2 1 Select Sample type DNA 50 for extinction coefficient 50 2 Buff top and bottom of pedestal to remove the nuclease free water To BLANK apply to the pedestal 1 5ul of elution buffer in which the nucleic acid is dissolved Click BLANK to calibrate the instrument 3 1 To make sure the blank is calibrated properly apply 1 51 of Elution buffer and click Measure Reading should be close to zero 4 Instrument is now ready to measure concentration of the nucleic acid sample Be sure to enter a Sample Name for each measurement 5 To quantify sample apply 1 5ul of the nucleic acid sample to the pedestal and click Measure 5 1 For Total RNA and aRNA 5 1 1 260 280 ratio of gt 2 0 5 1 1 1 Ifthe ratio is lower this may indicate the presence of protein phenol or other contami
56. ntrol click the Open Close Area Definition Control button Q shown on the right side of Figure 4 above the Scan Areas label in the software interface Figure 5 shows the components of the Area Definition Control 37 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 Load save area file Open Close Area Mie H Area Definition Control gt Q Show image gt Specify scan autogain Add colums and barcode areas H Areas T Arogan Asa T Barcode Avon D Siege Ame Mai Aron Link to Geometry gt Autogain Area Zoom function Slide Navigator Scan Area Barcode Area Add rows Figure 5 Components of the Area Definition Control b Ensure the following are selected Barcode area checkbox Autogain area checkbox if the Autogain checkbox was selected in the Parameter Control Figure 4 Single Area option this is the preferred option when scanning single and multiplex NimbleGen arrays For multiplex arrays you will use NimbleScan software s burst functionality to create individual image files for multiplex arrays c Review and if necessary adjust the scan area For NimbleGen 2 1M 3x720K and 12x135K arrays use the default selection for the scan area Optional For NimbleGen 385K and 4x72K arrays reduce the scan area which will reduce the scan time To define the scan area download and use area files availabl
57. rase the double stranded cDNA is modified to generate the final product blunt end double stranded cDNA ds cDNA At the completion of the procedure use of a spectrophotometer and microfluidic capillary electrophoresis assesses the quality of the purified ds cDNA Precautions Laboratory safety It is assumed that users have a sound knowledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Prevent excessive Rnase exposure As per all steps involved in RNA amplification it is essential to avoid latex glove Use only powder free nitrile gloves It is likewise important to guard against sources of dust and nucleases We recommend using Dnase and Rnase free plastics including barrier pipette tips In addition decontaminate both workspaces and pipettes with RNase Zap according to manufacturer s instructions Reverse Transcription to Synthesize First Strand CDNA Instrument Setup 1 Program MasterCycler Thermocycler 1 1 70 C for 10 minutes lid temperature 105 C 1 2 42 C for 60 minutes lid temperature 50 C Reagent Setup 1 Prepare 10mM dNTP mix 1 1 Combine 100ul of each dNTP dATP
58. rocentrifuge tube for First Strand Master Mix 2 2 Labeled 0 2ml thin walled PCR tube for each sample All centrifugation steps are at room temperature unless otherwise noted Remove the Total RNA samples from 80 C storage Keep on dry ice until ready to proceed with RNA amplification Procedure 1 Prepare first strand RNA primer mixture in a 0 2ml PCR tube Component Amount Total RNA 0 25ug 1 5ug Primer 1 0ul Nuclease Free water To final volume Total Volume 12 0ul 2 Incubate RNA Primer mixture for 10 minutes at 70 C Immediately remove the RNA primer mixture from the thermocycler and chill for 2 minutes in an ice bath Centrifuge briefly Keep on ice MasterCycler 70 C 10min 3 At room temperature prepare first strand master mix in a 1 5ml microcentrifuge tube labeled First Strand Master Mix Component Hrovided in kit Amountdplus 0 05 for part 2 error 10X First Strand Buffer 2 10ul dNTP Mix 4 20ul RNase Inhibitor 1 05ul ArrayScript 1 05ul Total Volume 8 40ul Gently pipette the First Strand Master Mix 4 times and flick 3 times to mix Centrifuge briefly Keep at room temperature Transfer RNA primer mixture to room temperature Add 8ul first stand master mix to each tube 20u1 total volume Gently pipette the reaction 4 times and flick 3 times to mix Centrifuge briefly Incubate for 2 hours at 42 C lid temperature 50 C
59. rray of the slide and each array designation for each burst image is appended to the original file name 1 3 Example Barcode A01_wavelength tif 1 4 Repeat steps 1 for each set of multiplex images of different designs 2 Select FILE gt OPEN The open an alignment dialog box appears Select a single array image Select the design file ndf Select the gene description file if available Click OPEN 3 Once the image has loaded the data is ready to match signal intensities with probe locations Select the AUTO ALIGN TOOL Ctrl A Zoom into the top corner of the array Ctrl G then Ctrl 7 Verify all fiducial controls line up correctly with the grid by shifting to each corner Ctrl 9 Ctrl 3 and Ctrl 1 4 Click FILE gt SAVE to save the gridded image 5 Confirm Experimental Integrity STC report for multiplex array formats only Select ANALYZE gt SAMPLE TRACKING Click ADD FILES Browse to select all 532nm gridded images of the multiplex array to be analyzed Click ADD TO LIST Click BROWSE to select destination of output file Save outfile with extension xls to review the STC report in MicroSoft Excel Open the file Ensure that a PRESENT call is reported for only the unique STC added to the hybridization reaction If more than one PRESENT call is report there may be contamination between hybridizations 42 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated
60. signment can be switched if desired Component Test Reference ds cDNA 0 6ug 0 6ug Diluted Cy3 Random Nonamers 16 8ul Diluted Cy5 Random Nonamers 16 8ul Nuclease free water To volume 321 To volume 321 Total 32 0ul 32 0ul 16 Incubate cDNA primer mixture for 10 minutes at 98 C MasterCycler 98 C 10min 17 Remove the reaction from the thermocycler Immediately transfer to an ice water bath and chill for 10 minutes Centrifuge briefly Return to ice bath 18 Prepare the Klenow master mix on ice in 1 5ml microcentrifuge tube labeled Klenow Master Mix Component er N 10mM dNTP Mix 4 2ul Nuclease Free water 3 36ul Klenow Fragment 3 5 exo 50U ul 0 84ul Total 8 4ul 19 Add 8ul Klenow master mix to reaction 40ul total volume Gently pipette the reaction 10 times to mix Centrifuge briefly Component Amount dNTP Klenow Mix 8 0ul Total 40 0ul 20 Incubate for 2 hours at 37 C lid temperature 105 C MasterCycler 37 C 2hr 21 During incubation add 44ul of Isopropanol to labeled 1 5ml microcentrifuge tube and then set aside 24 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 22 Remove the reaction from the thermocycler Centrifuge briefly 23 Add 4 0ul of Stop Solution 0 5M EDTA pH 8 0 to the reaction Compon
61. ssembly from the PMAT Place the Mixer Slide assembly on a 42 C heating block for 1 minute to facilitate adhesion of the mixer to the slide 8 Rub the Mixer Brayer over the mixer with moderate evenly distributed pressure to adhere the mixer and remove any bubbles between the adhesive and slide s surface For X1 mixers start in the center of the array and rub outwards For X4 and HX12 mixers first use a corner of the Mixer Brayer to rub the borders between the arrays and then rub outwards over the slide The adhesive will become clear when fully adhered to the slide s surface 9 Place the Mixer Slide assembly in the hybridization bay of the Hybridization System Load and hybridize samples to array Instrument Setup 1 Microman Capillary Piston Pipette CP10 or CP100 29 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 2 Microman Capillary Piston Pipette Tips CP10 or CP100 Sample Preparation Setup 1 2 3 4 NimbleChip Microarray Mixer Slide Assembly Mixer Seals Forceps KimWipes Procedure 1 At this point the hybridization reaction and Mixer Slide assembly are ready for sample loading Using the appropriate Gilson Microman pipette and tip draw up the designated loading volume inspect the pipette tip for bubbles Discharge and reload the pipette tip if there is a bubble It is recommended to have residual hybridization reaction in the tube to avo
62. t the parameters to use when scanning a To open the Parameter Control if not displayed go to the row of a slide to scan in the Magazine Control and click its Open Parameter Control button to display the Parameter Control b Do not adjust the following default parameters e Slide type e Channel 1 and Channel 2 e Laser Intensity 1 and Laser Intensity 2 e for slide nr 2 cycle 1 only c Adjust the following default parameters if necessary e Resolution e Speed Sensitivity e Autogain d To save any changes to the settings click Apply e Optional Click Save to save settings to a parameters file which allows the settings to be applied to other slides Optional To process the slide multiple times using the same or different parameters create up to 12 cycles To create a cycle close the Parameter Control by clicking the Close Parameter Control button Go to the slide row in the Magazine Control right click and select Add Cycle Open the Parameter Control Then specify and save parameters for the cycle as described above 8b 8e 9 Use the File Settings Control Figure 4 to specify image file settings 36 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 a To open the File Settings Control if not displayed go to the row of a slide to scan in the Magazine Control and click its Open Parameter Control button Figure 4 In the Parameter Contro
63. tes to warm plus 1 5 minutes per well measurement At the completion of the Chip Run clean the instrument and dispose of the used chip from the run 2 1 Open the lid and remove the used chip from the Agilent 2100 Bioanalyzer Place the electrode cleanser with fresh nuclease free water into the Agilent 2100 Bioanalyzer Close the lid and leave it for 10 seconds Open the lid and remove the electrode cleanser chip from the Agilent 2100 Bioanalzyer Keep the lid open for 10 seconds then close the lid Results Interpretations 1 Moderate to High Quality Total RNA Profile 1 1 Distinct 18S and 28S peaks 1 2 Low noise between peaks 1 3 Minimal low molecular weight contamination 1 4 Minimal high molecular weight contamination 1 5 High molecular weight contamination may indicate the presence of a contaminant i e DNA 50 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 200 150 100 50 25 200 500 1000 2000 4000 Figure 1 Electrophrogram of Total RNA nt 51 CGB Technical Report 2010 01 Microarray Protocol for High Throughput Genomics Last updated December 16 2010 2 High Quality amplified RNA aRNA Profile 2 1 One marker peak 2 2 Broad distribution medial molecular weight molecules 2 3 Minimal low molecular weight molecules 2 4 Minimal ribosomal RNA contamination lt 5 25 200 1000 4000 nt Figure 2 Electrop
64. tocol for High Throughput Genomics Last updated December 16 2010 Data Processing of Images Introduction NimbleScan Software allows for automated gridding of microarray image files to produce quantifiable data for analysis The protocol described here was adapted from Roche NimbleGen User s Guide for Expression analysis Briefly a tif image is uploaded into NimbleScan along with NimbleGen design files which describe both probe identities and locations to generate a tab delimited report with probe identities and feature signal intensities At the completion of the procedure data is presented in an appropriate format for normalization and comparative analysis Precautions Prevent loss of data After raw data is saved it is recommended to copy the files to a secondary independent data storage device or location Instrument Setup 1 Launch NimbleScan 2 6 Software Procedure 1 Ifthe slide image is a multiplex array 4x72K or 12x135K follow the steps below to separate each subarray into a separate image file Otherwise proceed to step 2 1 1 Select FILE gt BURST MULTIPLEX IMAGE The burst image dialog box appears To choose the tif files click ADD IMAGES Select the slide image of the same design Click ADD TO BATCH Browse to select the correct multiplex description file ncd Browse to select the desired output file destination in the OUTPUT BURST IMAGES TO field 1 2 Click BURST NimbleScan creates one file for each a
65. wledge of molecular biology techniques and safe laboratory practices Before undertaking a new protocol or using unfamiliar reagents users should review relevant Material Safety Data Sheets to identify potential hazards and recommended precautions For background in general molecular biology please see Molecular Cloning A Laboratory Manual J Sambrook and D W Russell Cold Spring Harbor Laboratory Press Prevent excessive light exposure Hybridized Cy labeled cDNA is light sensitive and should not be exposed to any unnecessary light When working with this reagent turn off all lights and close the blinds to the windows Prevent excessive ozone and humidity exposure Minimize ozone exposure and increased humidity levels as this leads to oxidation of the cyanine dyes Use of desiccant materials or a dehumidifier should be considered Prevent loss of data After image is saved it is recommended to copy the files to a secondary independent data storage device or location Instrument Setup 1 NimbleGen MS 200 Microarray Scanner 2 NimbleGen MS 200 Data Collection Software Procedure Start the control unit and log into your user account as msOperator or other account as set up by your system administrators Account msOperator Password 1 msOperator Turn on the scanner using the power switch on the left side Insert slides into the Slide Magazine as described in Figure 1 Numbered slots provide spacing for inserting slides 34 CGB
66. ybridization Array Format 4x72K Array 12x135K Array 385K Array Mixer X4 Mixer HX12 Mixer X1 Mixer 2 Position the Precision Mixer Alignment Tool PMAT so the hinge is on the left and then open it 3 Snap the mixer onto the two alignment pins on the lid of the PMAT with the tab end of the mixer toward the inside hinge and the mixer s adhesive gasket facing outward 4 Carefully unpack the slide and set aside its blue cassette for use at a later time While pushing back the PMAT s plastic spring with your thumb place the slide in the base of the PMAT so that the barcode is on the right farthest from the PMAT hinge and the corner of the slide sits against the plastic spring The NimbleGen logo and barcode number should be readable Remove your thumb and make sure the spring is pushing the corner of the slide so that the entire slide is touching the edge of the PMAT closest to you In addition be sure that the slide is lying flat against the PMAT 5 Using forceps remove the protective backing from the mixer s adhesive and close the PMAT so that the mixer s adhesive makes contact with the slide 6 Lift the lid by grasping the long edges of the PMAT while simultaneously applying pressure with a finger through the window in the lid of the PMAT to free the Mixer Slide assembly from the pins of the PMAT Be careful to avoid the arrays when applying pressure through the window 7 Remove the Mixer Slide a

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