SeraMir - System Biosciences
Contents
1. Aliquot 5ul of Mastermix into every well in your 384 well qPCR Plate 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 1ul 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 well A1 upper left into the Real time qPCR instrument and perform analysis run HELP 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 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual 2 Real time q2. SeraMir SBI Exosome RNA Amplification System Biosciences SeraMir Exosome RNA ver 6 Amplification Kit Cat RA800A TC 1 Cat RA805A 1 RA808A 1 Cat RA806A TC 1 Cat RA810A TC 1 Cat RA820A TC 1 Cat 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 sss 2 B Protocols sss 5 C Technical Supports 12 ll Licensing and Warranty Statement sss 13 Precipitate Exosomes from Serum with ExoQuick ExoQuick Exosomes Isolated from Serum qr Quick Optimized for serum Isolate Exosomes from Culture Media and Urine with ExoQuick TC ExoQuick TC I 4 N Es Fast Exoseme Isolation fromm Media and Urine 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI List of Components RAS00A TC 1 Components Amount ExoQuick for 3004 1 Smo ExoQuick TC for 800TC 1 om WashBuffer O O Om O SeraMir RNA Columns RAB004 1 SeraMir RNA Columns RASOOTC 1 Sx polyA Buffer oul MinC 25mM solution 20 ul SeraMir 3 Adaptor oligo PolyA polymerase oul 20u SeraMir SX RT Mix SeraMir Reverse Trans
3. sizes will range from 80 bases to as long as 1kb The SeraMir adaptors add 62 bases to the sizes of M E gt Wer the exoRNAs thus a base T7 IVT product corresponds to an exoRNAs insert sequence of about 20 bases 100 bp Amplified sense strand exoRNA 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 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual ll 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 purcha
4. 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 5 2011 11 23 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 SeraMirexoRNA method Phenol free Exosome Lysis PO oyy gt ExoQuick exosome pellets BIND NN WU VW Y SeraMir WASH columns ELUTE Phenol Trizol SeraMir Methods Technology AS itemi SeraMir Exosome RNA Amplification 3 l E a a ah 4 Hh x Y O A TEARS SN NI 4 i yali hey iG i My 4 ni A RO AE YA ue rn i Y DAPR sos i i de 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 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 Bioscie
5. 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 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 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual II EXOSOME RNA cDNA SYNTHESIS a Poly A reaction 5 ul eluted from spin exoRNA column Incubate at 37 C for 30 minutes 2 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 3 RT Reaction polyA exoRNA 10 ul from above 5X RT Master Mix 5 SeraMir Switch Oligo a Water PA 20 pl TOTAL Incubate at 42 C for 30 minutes Incubate at 95 C for 10 minutes HOLD at 15 C lil qPCR PROFILING OF exo cDNA cat RASO5A 1 SeraMir Spike in RNA qPCR assay and RA810A 1 SeraMir Exosome RNA 384 microRNA gPCR 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 exo cDNA 2X SYBR Master Mix
6. 5 SeraMir Spike in assay primer SeraMir 3 Reverse qPCR primer 0 5 ul from above 15 ul 1 ul 0 5 ul 13 ul 30 uI TOTAL Page 6 ver 5 2011 11 23 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 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 For 384 well plates Add Perwell 1 pl of 1 50 dilution SeraMir 3 Reverse qPCR primer Water A i SBI recommends Fermentas 2X Maxima SYBR Green ROX qPCR Master Mix cat K0221 Page 8 ver 5 2011 11 23 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 Sample qPCR data for the SeraMir Spike in RNA Amplification Plot E SeraMir Exosome RNA Amplification 1 10 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 SeraMir 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 ul 2X SYBR Green qPCR Mastermix buffer 39 ul 3 SeraMir Reverse Primer 10M Sul User synthesized SeraMir cDNA 1090 ul Nuclease free water 2284 ul Total
7. PCR 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 Create a detector 1 Create a new Detector Detector Manager rial fer Fiter Settings 2 Name the Detector ae E ux Reporter Quencher Color Madcation Date Owner Creston Date any name will do ete Cancer SY SYER Non Fluorescent MR Tur Mer 05 10 31 20 PST 2007 Tue Mar 06 10 31 20 PST 2007 3 Select Reporter Dye as SYBR Green 4 Select Quencher Dye as none Instrument setup qPCR cycling and sete trans data accumulation gems Gear coca conditions 4 Sod rma 7 3000 Emden ademas te mai Profle Jats increment Reep Fate Daia Cokectorn ata sj Standard Protocol fapana 1 50 C 2 min 2 95 C 10 min 3 95 C 15 sec 4 60 C 1 min 40 cycles of Stage 3 data read at 60 C 1 min Step l 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
8. criptase SeraMir 2x Tag PCR Mix 250w SeraMir PCR amplification primer mix 10 ml 20 mi 50 ATP solution SmM 30 ul polymeras 10 ul 10 ul 20 ul F SeraMir Reverse q PCR primer RA20541 Components Control spike in small RNA Control spike in small RNA q 3 SeraMir Reverse g PCR pri RAS06A TC 1 Components Amount ExoQuick for RABOGA 1 ExoQuick TC for RABOGTC 1 Lysis Buffer aml SeraMir RNA Columns R43064 1 SeraMir RNA Columns RASOSTC 1 RA308A 1 Components Amount Lysis Buffer am Wash Buffer Elution buffer arahi j Te 0 1 rey Mk fm LT Amount 384 well SeraMir Profiler 20 profiles 3 SeraMir Reverse gPCR primer RA8204 1 Components Amount All of RABOOA 1 or BOOTC 1 All of RASOSA 1 All of RAS1OA 1 RA amp 820TC 1 Components Amount All of SOOTC and all of BOGTC 1 but with E A 20 reactions 50 ml ExoQuick TC All of RASOSA 1 All ofRAS104 1 20 profiles Page 2 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 4C Please check each box for proper storage conditions upon receipt Properly stored kits are stable for 1 year from the date received 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
9. d cell debris Thaw serum sample on ice Combine 500ul serum 120 ul ExoQuick Or 1 ml ExoQuick TC with 5 ml Media Mix well by inversion three times Place at 4 C for 30 minutes serum or for 6 hours to overnight urine media Exosome Isolation and Lysis 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 cat RA805A 7 9 Add 200ul of 100 Ethanol vortex 10 seconds 10 Assemble spin column and collection tube 11 Transfer all 600ul 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 rom 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 i Discard collection tube and assemble exoRNA spin column with a fresh Elution RNase free 1 5ml elution tube not provided Add 30ul ELUTION Buffer directly to membrane in spin column Centrifuge at 2 000 rpm for 2 minutes loads buffer in membrane Increase speed to 13 000 rpm and centrifuge for 1 minute elutes exoRNAs You should have recovered 30
10. m sample PCR IVT Amplify N M Un Amp Tail and Tag exoRNA ExoQuick exosome gt precipitation Supernatant Exosome pellet gt x EM image 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI PROTOCOL AT A GLIMPSE Precipitate serum exosomes and purify exoRNAs Serum a Simple one step Phenol free 2 precipitation Exosome Lysis om Lysate wn BIND 3 minute clean up ms ELUTE sen Mia sem pellet Exosomal RNAs SSB 5 3 Ss gt a 3 5 y F E 5 3 Tail exoRNAs and synthesize double tagged cDNA Exosomal RNAs s 3 Sw 3 AAAAAAAAAA Polyadenylate Pone N 5 Switch swawwwwAAAAAAAAAA Captor 3 Adaptor Adaptor 5 GGG 3 Reverse Transcription 5 switch and tag ms swwwwwwwAAAAAAAAAA 3 Adaptor AE SD First strand cDNA q PCR ready SSC 5 adaptor 5 primer gt 3 JCCcc 5 Amplification for 3 adaptor second strand cDNA primer T7 IVT ready 5 eG y 3 CCC 5 PCR Amplify PCR IVT Amplify M Un Amp M Un Amp 500 bp Exosomal RNA cDNA 200 bp 100 bp Amplified exoRNA User Manual Page 4 ver 5 2011 11 23 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 B Protocol I EXOSOME RNA ISOLATION PROTOCOL FROM 500ul SERUM or 5ml Media Collect biofluid and centrifuge at 3000 x g for 15 minutes to remove cells an
11. nces SBI User Manual IV EXOSOME cDNA AMPLIFICATION The first strand exoRNAs cDNA made in STEP II 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 Per reaction ET amplified Primer Mix Water AB 25 ul TOTAL Place the reactions in a thermal cycler and cycle using the following program e 95 C for 5 min e 95 C for 25 sec e 60 C for 20sec 35 Cycles e 72 C for 30 sec e 72 C for 30 sec e 15 C hold Visualize the PCR products on a 1 5 agarose gel load 5 ul per well PCR Amplify Exosomal RNA cDNA v EXOSOME SENSE RNA AMPLIFICATION SBI recommends using Epicentre s AmpliScribe 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 ul 100 mM ATP F 1 8 ul 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 ul Total reaction volume Incubate at 45 C for 45 minutes Page 12 ver 5 2011 11 23 www systembio com SeraMir Exosome RNA Amplification Cat RA800 805 806 808 810 820A TC 1 Visualize the PCR products on a 1 5 agarose gel load 5 ul per well T7 IVT Amplify 1 2 3 The RNA
12. need 100 Ethanol molecular grade ver 5 2011 11 23 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 SBI 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 ExoQuick TC media e Increase sensitivity amplify exoRNAs for qPCR e Gain more data use T7 IVT amplified sense exoRNAs for microarrays and NextGen sequencing Seru
13. of the Product SBs 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 Page 14 ver 5 2011 11 23 www systembio com
14. ser 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 disclaims 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
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