User Manual
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1. a Pretreatment of RNA sample to remove contaminated genomic DNA i Addthe following reagents to a PCR reaction tube for each RNA sample Components Volume RNA 0 1 2ug Tul 10 X Buffer 1 ul DNase 1 ul DNase RNase free ddH20 7 ul Total 10 pl ii Incubate in a thermal cycler at 42 C for 5 minutes then immediately put the tube on ice for at least 1 minute b Reverse transcription reaction i Prepare the cocktails following the table bellow for different sample numbers Reaction number Component 1X 2X 4X 8X DNase Rnase free ddH20 Bul 6 ul 12 ul 24 ul Oligo mix 1 ul 2 ul 4 ul 8 ul 5X buffer 4 ul 8 ul 16 ul 32 ul RT enzyme Mix 2 ul 4 ul 8 ul 16 ul Total 10g 20pl 40 pl 80 pl User s Manual qLight qPCR Gene Expression Assay System Mix well Add 10 ul of the cocktail to the each pretreatment reaction for each sample Gently mix incubate at 42 C for 20 minutes then followed by at 95 C for 5 minutes Add 95 ul DNase RNase free ddH20 to each reaction mix well and place it on ice until use Or store at 20 C 2 Assemble 96 well PCR array plate reactions amp run real time PCR plate To aclean reservoir add Component Volume DNase Rnase free ddH O 985 uL Targetgreen SYBR green qPCR Master Mix 1100 uL Diluted reverse transcription product 115 uL Total 2200 uL Mix well add 20 ul of the mix to each well of the array plate using a multi channel pipette Seal the well with the provided trans2. One tube contains 400 ul primer mixes enough for 200 reactions Shipping condition cold with blue ice Storage condition 4 9C for 6 months Reference amp QC Array Plate Kit optional as an option for quality control with internal reference reactions and positive QC control reactions array to be used with a Forcustom array kits with target gene number less than 49 b Forsingle gene assay primer mix kit Shipping condition Ambient temperature Storage condition 4 9C for 6 months User s Manual qLight qPCR Gene Expression Assay System 3 Additional Materials Required Note The following reagents are mandatory for optimal results The counterparts or equivalent kits from other vendors are not recommended due to unique features of our system design 1 qLight Genomic DNA free First Strand cDNA Synthesis Kit Cat RTO3 2 qLight SYBR green qPCR Master Mix refer to the table below for the Master mix products for your PCR cycler Master mix is designed based on the reference dye per manufacturers of common PCR cyclers Master Mix Type Cat No Size Real Time PCR Cyclers 2X 96 well plates 200 MR1 02 rxn 2X1 3mL All ABI amp Stratagene qLlight ROX Instrumentation Eppendorf SYBR green 12X 96 well plates 200 MR1 12 Mastercycler ep realplex Master Mix rxn 12X1 3mL Instruments with ROX filter MR1 24X 96 well plates 200 set MR1 24 rxn 24X1 3mL 2X 96 well plates 200 MF2 02 x m
3. 00000000 1000000000000 Sae WARS AY o0 D y d I es S XS estes LEE Fig 2 A typical gene expression signature array plate layout Data analysis template is developed based on AACt method based on the fold changes in gene expression between your test samples and control samples Following simple steps below the relative expression level fold change is automatically computed between two samples for comparison 1 Download the analysis template from our website link here 2 Enter the raw Ct numbers from the PCR reaction plates into the corresponding rows and columns of the analysis template by simple copying and pasting following the instruction described in the instruction sheet 3 The RT positive control reaction RTPC and PCR positive reaction control reaction PCRPC results are displayed in the QC control sheet 4 Thetarget gene results will be automatically displayed in result sheet Two fold changes are built in default positive changes User s Manual qLight qPCR Gene Expression Assay System 5 Protocol Note The RT kit and the SYBR green master mix are mandatory for desired performance Other commercial equivalent products are not expected to work due to the unique designs of primer sets and the control layout 1 cDNA synthesis using qLight Genomic DNA free First Strand cDNA Synthesis Kit Cat RT 01 Note The protocol and reagents are not compatible with any counterpart products from other vendors
4. The second Generation of PCR Array Tw Z PCRarray 2 0 lt 46400 Benedict Drive Ste 105 Sterling VA 20164 P 1 866 489 5499 F 1 703 637 9863 support pcrarray2 com www pcrarray2 com qLight qPCR Gene Expression Assay System Simple Fast amp Reliable Version 1 2 For Research Use Only Not For Use in Diagnostics User s Manual qLight qPCR Gene Expression Assay System 1 Table of Contents 1s Table ot Contents oerte rn e P te n A eee recede here ee 2 Materials Provided 5 reet ete reiten ne eee seed tt erede e vt ees 3 Additional Materials Required esses esee eee enne nhi nnnn nnns ennt asas sse nee tasa sanis As IntKOdUCctloh orte reb ree retta e eec tote ER repre e EM e Fete e echar E RE Regn 5 DEOLOCOlL os tet trt oem ttti ai dies emus st rires User s Manual qLight qPCR Gene Expression Assay System 2 Materials Provided Validated primer mix in array plate or in tubes 1 2 3 4 Pre designed array qPCR kit 96 well qPCR array plates with sets of PCR primers for up to 84 target genes you have ordered Shipping condition Ambient temperature Storage condition 4 9C for 6 months Custom array qPCR Kit 96 well qPCR array plates with sets of PCR primers per your request Shipping condition Ambient temperature Storage condition 4 9C for 6 months Single gene qPCR kit Primer mix solutions in 1 5ml microtube s for the gene of your interest
5. b qlight rxn 3m Fluorescein SYBR TP 12X 96 well plates 200 BioRad iCylcer MyiQ and Green Master rxn 12X1 3mL iQ5 Instrumentation Mix MF2 24X 96 well plates 200 MF2 24 rxn 24X1 3mL TE MN3 02 REWE plates 200 BioRad Opticon Opticon 2 amp i ja PROPRIE Chromo 4 Roche Green Master A 12X 96 well plates 200 LightCycler 480 System Mix MM3 MN3 12 rxn 12X1 3mL Eppendorf Mastercycler ep No reference realplex Instruments without dye 24X 96 well plates 200 x MN3 24 ROX filter set rxn 24X1 3mL User s Manual qLight qPCR Gene Expression Assay System 4 Introduction qLight gene expression qPCR assay system is a simple fast and reliable real time PCR system It has three major product components with compatible design to assuare quality and performance with convenience These components are reliable templates using our genomic DNA gDNA free first strand cDNA synthesis system performance validated primers designed with TG primer design software with genome wide design capacity and qLight SYBR green real time PCR Master mix Following products are offered with qLight gene expression qPCR assay system 1 Catalogued qLight pathway qPCR array gene expression profiling kit e Sets of designed primers for pre selected pathway genes up to 84 genes dried down to 96 well plate e With internal reference reactions and external positive reactions monitoring RT reaction and PCR reaction e S
6. imply add the master mix and cDNA cocktail for the PCR reactions 2 Custom qPCR array gene expression profiling kit e The flexibility to design your array with benefits of our manufacturing consistency and internal reference and QC standard e Simply add the master mix and cDNA cocktail to your custom qPCR array plates see page for array layout and the protocol 3 Single gene expression qPCR assay kit e Convenience to perform single gene expression assays see page for the protocol 4 Reference and QC Array Benefit e nternal reference reactions and external control reactions e To be used with customer qPCR array products and single gene assays e Eight samples can be analyzed simultaneously see page and page Simple genomic DNA free first strand cDNA synthesis to eliminate contamination concern of genomic DNA High PCR performance with validated primers and matched PCR Master mix for your cycler Built in controls to monitor quality and performance Simple amp Fast procedure to profile up to 84 gene expression in 2 5 hours Data analysis template offered for you to obtain your results within minutes User s Manual qLight qPCR Gene Expression Assay System The simple three step procedure See Fig 1 total time 3 hours Step 1 First strand cDNA synthesis in 35 minutes from RNA to cDNA Step2 Setup PCR reaction in 10 15 minutes i Prepare PCR cocktail by mixing synthesized cDNA with qLight qPCR master mi
7. parent film or the 8 cap strip Spin briefly to collect the mixture solution to the well bottom Place the plate on PCR cycler and run the reaction using the cycling program below Initial denaturation amp Taq activation stage Cycling stage Denaturation Annealing amp extension Dissociation curve Melting curve stage Temperature Time 95 9c 10 min 96 C 15 sec 60 C 1 min Add the stage following cyclers instruction Cycling number User s Manual qLight qPCR Gene Expression Assay System 3 Data analysis a Export raw Ct values from your PCR run file after the setup of the threshold and baseline as below i Setup threshold at 0 2 ii Set up baseline up the starting at cycle 3 and stop at cycle 15 b Download software template fitting to these array layout are available our website c Follow the instruction of data analysis template for your array layout enter the Ct values of the target gene array plate run along with those of reference reaction and QC control array plate run 10
8. x ii dispense the cocktail to the selected qLight array plate Step3 Runreal time PCR reaction in about two hours First strand cDNA synthesis Remove genomic DNA from RNA sample Synthesize first strand cDNA from DNA free RNA sample Run real time PCR reaction Prepare cocktail with cDNA and Ce 2X SYBR green PCR mastermix LE FJJ CH goo MOON Q i ili GO000SG00 000 Loading PCR cocktail into 96 Sod d Odd Odd ti ak ale ck well real time PCR plate Edbeie debeo dicet 299900000009 Run PCR reactions in real time PCR cycler and analyze the data Detectable Detectable ILLI C Van Fig 1 Array profiling procedure flowchart Array plate layout and data analysis A typical array plate contains following components See Fig 2 1 Wells A1 to G12 Reactions of 84 target genes to be analyzed in your samples 2 Wells H1 to H8 Reactions of 4 housekeeping genes in duplicate as internal reference controls 3 Wells H9 and H10 5 and 3 portion reactions for same housekeeping gene to monitor if input RNA sample is integrate User s Manual qLight qPCR Gene Expression Assay System 4 Well 11 external reverse transcription positive control reaction RTPC to monitor RT reaction of input smaples 5 Well 12 external PCR positive control reaction PCRPC to monitor the PCR reaction 123 4 5 67 8 9 10 11 12 0O00000000000 s 00000000000 cO00000000099 1200009990000 OOO0O00000099909 r 209990000009 co000
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