SeraMir Protocol and User Manual
Contents
1. 820A TC 1 Il qPCR PROFILING OF exo cDNA cat RA805A 1 SeraMir Spike in RNA qPCR assay and RA810A 1 SeraMir Exosome RNA 384 microRNA qPCR Profiler To test your exo cDNA we recommend performing a qPCR assay for the RA805A 1 Spike in RNA control or proceed to the 384 well SeraMir Profiler setup qPCR array contains the Spike in qPCR assay For 96 well plates Add Per well exo cDNA 0 5 ul from above 2X SYBR Masier Mix 15 ul 5 SeraMir Spike in assay primer 1 ul SeraMir 3 Reverse qPCR primer 0 5 ul Water 13 ul 30 pl TOTAL SBI recommends Fermentas 2X Maxima SYBR Green ROX qPCR Master Mix cat K0221 For 384 well plates Add Per well exo cDNA 1 ul of 1 50 dilution 2X SYBR Master Mix 3 ul 5 SeraMir Spike in assay primer 0 2 ul SeraMir 3 Reverse qPCR primer 0 1 ul Water 1 7 ul 6 ul TOTAL SBI recommends Fermentas 2X Maxima SYBR Green ROX qPCR Master Mix cat K0221 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Sample qPCR data for the SeraMir Spike in RNA Amplification Plot Dissociation Analysis PEPENE p Argifsculiun Piol SeraMir Exosome RNA Amplification Tm 76 C If 5 ul of the SeraMir Spike in RNA was used during the exoRNAs isolation then you should expect to observe a Ct of about 15 to 20 Setup of the 384 well SeraMir2. T7 Flash Transcription Kit catalog ASF3257 4 3 ul RNase Free water 2 ul Amplified exo cDNA from STEP IV 2 ul AmpliScribe T7 Flash 10X Reaction Buffer 1 8 pl 100 mM ATP 1 8 pl 100 mM CTP 1 8 ul 100 mM GTP 1 8 ul 100 mM UTP 2 ul 100 mM DTT 0 5 ul RiboGuard RNase Inhibitor 2 ul AmpliScribe T7 Flash Enzyme Solution 20 yl Total reaction volume Incubate at 45 C for 45 minutes Visualize the PCR products on a 1 596 agarose gel load 5 ul per well T7 IVT Amplify 1 2 3 100bp Amplified sense strand exoRNA 888 266 5066 Toll Free 650 968 2200 outside US The RNA sizes will range from 80 bases to as long as 1kb The SeraMir adaptors add 62 bases to the sizes of the exoRNAs thus a base T7 IVT product corresponds to an exoRNaAs insert sequence of about 20 bases Page 13 System Biosciences SBI User Manual C Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com Page 14 ver 11 2014 06 027 www systembio com SeraMir Exosome R
3. 384 Profiler cat RA810A 1 The phenol free exosome lysis step coupled to the small RNA binding columns isolates exoRNAs with much higher purity than Trizol Phenol based methods The SeraMir exoRNAs are compatible with downstream polyadenylation and reverse trancription reactions for amplification and accurate qPCR profiling 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual IV EXOSOME cDNA AMPLIFICATION The first strand exoRNAs cDNA made in STEP Il Can be amplified to make double stranded exo cDNA compatible with T7 in vitro transcription reactions to make amplified sense RNAs that work with most microRNA microarrays and can be adapted to use with NextGen sequencing preparation protocols Add exoRNA amplified cDNA 2X PCR Master Mix 12 5 ul 5 SeraMir PCR 2 ul from above Primer Mix 1 ul Water 9 5 ul 25 ul TOTAL Place the reactions in a thermal cycler and cycle using the following program 95 C for 5 min 95 C for 25 sec 60 C for20sec 35 Cycles 72 C for 30 sec 72 C for 30 sec 15 C hold Visualize the PCR products on a 1 596 agarose gel load 5 ul per well PCR Amplify Amp Exosomal RNA cDNA Page 12 ver 11 2014 06 027 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 V EXOSOME SENSE RNA AMPLIFICATION SBI recommends using Epicentre s AmpliScribe
4. z Thermal Cycer Protocol 7 Stendard 9500 Emulation Sample Volume ey fio Ta A Auto neremert Rane Rate Data Cotectin Stage 1 Stage 2 Stage 3 aj Standard Protocol Reposts 5 50 C 2 min 95 C 10 min 95 C 15 sec 60 C 1 min 40 cycles of Stage 3 data read at 60 C 1 min Step Bow Add Dissociation Stage An additional recommendation is to include a Dissociation Stage after the qPCR run to assess the Tm of the PCR amplicon to verify the specificity of the amplification reaction Refer to the User Manual for your specific instrument to conduct the melt analysis and the data analyses of the amplification plots and Cycle Threshold Ct calculations In general Cycle thresholds should be set within the exponential phase of the amplification plots with software automatic baseline settings Page 10 ver 11 2014 06 027 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 Sample 384 well SeraMir Profiler Data Serum RNA extraction methods SeraMir exoRNA method Phenol free Exosome Lysis m y ExoQuick exosome pellets BIND BA 222 SeraMir WASH columns ELUTE Phenol Trizol SeraMir Methods Technology SeraMir Exosome RNA Amplification The results are clear obtain more data with SeraMir Serum RNA prepared by conventional Trizol versus the SeraMir kit Profiling of 380 Human microRNAs across the SeraMir
5. EXOSOME RNA ISOLATION PROTOCOL FROM 500p SERUM or 5ml Media Collect biofluid and centrifuge at 3000 x g for 15 minutes to remove cells and cell debris aw serum sample on ice Combine 500yl serum 120 ul ExoQuick manly Or 1 ml ExoQuick TC with 5 ml Media and Lysis Mix well by inversion three times Place at 4 C for 30 minutes serum or 6h overnight urine or media Centrifuge at 13 000 rpm for 2 minutes Remove supernatant keep exosome pellet Add 350 ul LYSIS Buffer to exosome pellet and vortex 15 seconds Place at room temperature for 5 minutes to allow complete lysis optional add 5ul of SeraMir control RNA spike in 9 Add 200yl of 100 Ethanol vortex 10 seconds 10 Assemble spin column and collection tube 11 Transfer all 600g to spin column 12 Centrifuge at 13 000 rpm for 1 minute exoRNA check to see that all flowed through Purification otherwise spin longer 13 Discard flow through and place spin column back into collection tube 14 Add 400ul WASH Buffer 15 Centrifuge at 13 000 rpm for 1 minute 16 Repeat steps 13 to 15 once again total of 2 Washes 17 Discard flow through and centrifuge at 13 000 rpm for 2 minutes to dry IMPORTANT Discard collection tube and assemble spin column with a fresh RNase free 1 5ml elution tube not provided Add 30u ELUTION Buffer directly to membrane in spin column exoRNA Elution 20 Centrifuge at 2 000 rpm for 2 minutes loads buffer in membrane 21 Inc
6. NA Amplification Cat RA800 805 806 808 810 820A TC 1 Licensing and Warranty Statement Limited Use License Use of the SeraMir Exosome RNA Amplification Kit i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI discla
7. Profiler cat RA810A 1 Mastermix qPCR Reaction Set up for ONE entire 384 well qPCR plate To determine the expression profile for your miRNAs under study mix the following for 1 entire 384 qPCR plate For 1 entire plate 1150 pl 2X SYBR Green qPCR Mastermix buffer 39 ul 3 SeraMir Reverse Primer 10uM 5 ul User synthesized SeraMir cDNA 1090 ul Nuclease free water 2284 ul Total Aliquot 5ul of Mastermix into every well in your 384 well qPCR Plate Page 8 ver 11 2014 06 027 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 SBI has tested and recommends SYBR Green Master mix from three vendors 1 Fermentas 2X Maxima SYBR Green ROX qPCR Master Mix cat K0221 2 Power SYBR Master Mix Cat numbers 4368577 4367650 4367659 4368706 4368702 4368708 4367660 from Applied Biosystems 3 SYBR GreenER qPCR SuperMix for ABI PRISM instrument from Invitrogen Cat numbers 11760 100 11760 500 and 11760 02K Resuspend the MicroRNA assay Primers with 22ul water in each well before use Spin briefly to collect contents at bottom of wells Then Load il per well of each of the Primers from the Primer Stock plate into your qPCR plate well A1 into qPCR plate A1 etc Once reagents are loaded into the wells cover the plate with an optical adhesive cover and spin briefly in a centrifuge to bring contents to bottom of wells Place plate in the correct orientation wel
8. SeraMir 5X RT Mastermix 80 ul SeraMir Reverse Transcriptase 20 ul SeraMir 2X Taq PCR Mix 250 ul PCR amplification primer mix 20 ul SeraMir 3 Reverse qPCR primer 600 ul RA805A 1 Components Amount Control spike in RNA control 100 ul Control spike in RNA qPCR assay 100 assays SeraMir 3 Reverse qPCR primer 600 ul RA806A TC 1 Components Amount ExoQuick for RA806A 1 5ml ExoQuick TC for RABO6TC 1 10 ml Lysis Buffer 8ml Wash Buffer 20 ml Elution buffer 750 ul SeraMir RNA Columns RA806A 1 20 columns SeraMir RNA Columns RA806TC 1 10 columns RA808A 1 Components Amount Lysis Buffer 8ml Wash Buffer 20 ml Elution buffer 750 ul SeraMir RNA Columns RA806A 1 20 columns RA810A 1 Components Amount 384 well SeraMir Profiler 20 profiles SeraMir 3 Reverse qPCR primer 600 ul RA820A 1 Components Amount All of RA800A 1 or 800TC 1 20 reactions All of RA805A 1 100 assays All of RA810A 1 20 profiles RA820TC 1 Components Amount All of 800TC and all of 806TC 1 but with 50 D reactions ml ExoQuick TC All of RA805A 1 100 assays All of RA810A 1 20 profiles Page 2 ver 11 2014 06 027 User Manual The SeraMir kits are shipped on blue ice 20 C especially for the cDNA synthesis reagents The RNA columns and buffers can be stored at room temperature and the ExoQuick or ExoQuick TC is stored at 4 C Please check each box for proper storage conditions upon receipt Properly stored kits are stable for 1 year from the date re
9. Spr amp SeraMir Exosome RNA Amplification System Biosciences SeraMir Exosome RNA Amplification Kit Cat RA800A TC 1 Cat RA805A 1 RA806A TC 1 Cat RA808A 1 Cat RA810A TC 1 RA811A TC 1 Cat RA820A TC 1 RA821A TC 1 User Manual See boxes for proper storage of the kit components upon receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 Contents Protocol A Overview LLL 2 B Protocols 4 C Technical Support sese 13 ll Licensing and Warranty Statement 14 eQuick Optimized for serum Fast Exosome Isolation frofmMedia and Urine 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI List of Components RA800A TC 1 Components Amount ExoQuick for 800A 1 5ml ExoQuick TC for 800TC 1 10ml Lysis Buffer 8ml Wash Buffer 20 ml Elution buffer 750 ul SeraMir RNA Columns RA800A 1 20 columns SeraMir RNA Columns RA800TC 1 10 columns 5x polyA polymerase Buffer 40 ul MnCl 25mM 20 ul ATP 5mM 30 ul PolyA polymerase 10 ul 3 Adaptor oligo 10uM 10ul 5 Switch oligo 10uM 20 ul
10. ceived The reaction size is based on using 500 ul serum with ExoQuick or for 5 ml media or urine using ExoQuick TC for exosome isolation and exoRNAs amplification You will need 100 Ethanol molecular grade www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 SeraMir Exosome RNA Amplification A Overview RNAs present in patient body fluids and cell culture media are a rich and untapped source of disease related biomarkers The RNAs are stable in serum because they are encapsulated in circulating exosomes Exosomes are 40 100 nm membrane vesicles secreted by most cell types in vivo and in vitro Exosomes are found in blood urine amniotic fluid malignant ascite fluids cell media and contain distinct subsets of microRNAs depending upon the tumor or tissue from which they are secreted The SeraMir kit includes everything needed to accurately and sensitively measure RNAs from serum samples Exosomes are efficiently isolated using SBl s ExoQuick solution and the exoRNAs are purified using a phenol free lysis buffer and rapid spin columns The SeraMir kit enables the 3 tailing and simultaneous tagging of both 5 and 3 ends during cDNA synthesis ready for qPCR Primers for PCR amplification are included for highly sensitive applications e No time consuming ultracentrifugation to isolate exosomes e Reduce variability isolate exosomes first with ExoQuick serum or Ex
11. ims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2011 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 15
12. l A1 upper left into the Real time qPCR instrument and perform analysis run Use a Multichannel pipette to load the qPCR plate with MasterMix and Primers Pour the Mastermix into a reservoir trough and use a 8 or 12 channel pipette to load the entire 384 well qPCR plate with the Mastermix Then load the primers from the primer plate to the qPCR plate using a separate multichannel pipette 2 Real time qPCR Instrument Parameters Follow the guidelines as detailed for your specific Real time instrumentation The following parameters tested by SBI were performed on an Applied Biosystems 7900HT Real time PCR System but can also apply to any other 384 well system The details of the thermal cycling conditions used in testing at SBI are below A screenshot from SBI s 7900HT Real time instrument setup is shown below also Default conditions are used throughout 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI Create a detector 1 Create a new Detector 2 Name the Detector any name will do 3 Select Reporter Dye as SYBR Green 4 Select Quencher Dye as none Instrument setup qPCR cycling and data accumulation conditions User Manual Detector Manager 2 ree resi Reporter Guencher Coor Medication Date Ower resin Date Cancer SYER EI Non Fucrescert il Tue Mar 05 10 31 20 PST 2007 Tue Mar 06 10 31 20 PST 2007 Setup etranert
13. oQuick TC media e Increase sensitivity amplify exoRNAs for qPCR e Gain more data use T7 IVT amplified sense exoRNAs for microarrays and NextGen sequencing Serum sample PCR IVT Amplify D Tail and Tag exoRNA ExoQuick exosome gt precipitation Supernatant Exosome pellet gt EM image PROTOCOL AT A GLIMPSE 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Precipitate serum exosomes and purify exoRNAs Serum G Simple one step Phenol free e a precipitation sh Exosome Lysis ar Lysate ai P mo 3 minute H exoRNA A XE Us ELUTE clean up Mrs sw ellet a Exosomal RNAs ta AE s 5 3 a MERE r 5 3 Tail exoRNAs and synthesize double tagged cDNA Exosomal RNAs m s e AAAAAAAAAA Polyadenylate 5 Switch sunm AAAAAAAAAA 3 Adaptor 3 Adaptor Adaptor 5 e GGG 3 Reverse Transcription s switch andtag pO sve AAAAAAAARA 3 Adaptor n A 50 UT t First strand cDNA qPCR ready 3 1ccc 5 5 adaptor primer l T O 7 Amplification for 3 adaptor second strand cDNA Q primer T7 IVT ready i cc z PCR Amplify PCRHVT Amplify M Un Amp Exosomal 500 bp RNA cDNA Amplified exoRNA Page 4 ver 11 2014 06 027 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 B Protocol l
14. rease speed to 13 000 rpm and centrifuge for 1 minute elutes exoRNAs 22 You should have recovered 30 40ul exosome RNA The yield of RNA from isolated exosomes is different depending on the starting biofluid or the type of cells that were grown in 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual culture Different cell types secrete varying levels of exosomes For serum the level of RNA isolated from 500 ul is usually in the 500ng range and can be measured using an Agilent Bioanalyzer or a NanoDrop Spectrophotometer The recovery from cell media varies depending the cell type and growth confluency Il EXOSOME RNA cDNA SYNTHESIS Poly A reaction Per reaction Add 5 ul eluted from spin exoRNA column 5X polyA Buffer 2 pl MnCl 25 mM Tul ATP 5 mM Tot polyA polymerase ot Incubate at 37 C for 30 minutes m Adaptor Anneal Reaction Add 0 5 ul SeraMir 3 Adaptor Oligo Incubate at 60 C for 5 minutes Incubate at Room temperature 2 minutes Place on ICE RT Reaction Add Per reaction polyA exoRNA 10 ul from above 5X RT Master Mix 4 ul 5 SeraMir Switch Oligo 1 pl Reverse Transcriptase 1 ul Water 4 ul 20 ul TOTAL Incubate at 42 C for 30 minutes Incubate at 95 C for 10 minutes HOLD at 15 C Page 6 ver 11 2014 06 027 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810
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