User`s Manual and Instructions
Contents
1. CEJ Biocnains wew biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com User s Manual and Instructions RapidSeq High Yield Directional mRNA Sample Prep Kit Catalog Number KS073012 KS073012 I KS073012 II KS073012 IIl KS073012 IV Introduction Analysis of differential RNA expression helps us to understand biological pathways and molecular mechanisms which are involved in the regulation of cell function individual development and disease progression Sequencing technologies provide a powerful tool for transcriptome analysis Next generation sequencing NGS has great advantages over conventional methods by tremendously reducing the sequencing costs and increasing genome coverage Transcriptome sequencing or RNA seq is a novel method for gene expression analysis Advantages of RNA Seq include no bias toward known RNA molecules as with probe based technologies ability to detect novel and rare transcripts or novel alternative splice isoforms and direct measurement of transcript abundance within biological samples The RapidSeq kit aims to prepare directional NGS libraries for subsequent cluster generation using purified mRNA as start material The protocol includes steps for mRNA fragmentation purification adapter ligation reverse transcription PCR amplification and DNA fragment enrichment to generate strand specific library product compatible with illumina NGS platform Figure 1 F 7532. 60 ul KS073012 9 RT Enzyme 60 ul KS073012 10 Blue Universal Primer 60 ul KS073012 11 PCR MasterMix 700 ul KS073012 12 F 753 3UMRevD KS073012UD Active Date 11012013 CE J BioChain www biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Table 3 Contents List of RapidSeq High Yield Directional mRNA Sample Prep Kit Box 3 of 3 Store at 20 C KS072012 1 Item Amount in kit ul Part No Sequence Aligner 1 10 KS072012 1 ATCACG Aligner 2 10 KS072012 2 CGATGT Aligner 3 10 KS072012 3 TTAGGC Aligner 4 10 KS072012 4 TGACCA Aligner 5 10 KS072012 5 ACAGTG Aligner 6 10 KS072012 6 GCCAAT Aligner 7 10 KS072012 7 CAGATC Aligner 8 10 KS072012 8 ACTTGA Aligner 9 10 KS072012 9 GATCAG Aligner 10 10 KS072012 10 TAGCTT Aligner 11 10 KS072012 11 GGCTAC Aligner 12 10 KS072012 12 CTTGTA KS071012 II Item Amount in kit pl Part No Sequence Aligner 13 10 KS072012 13 AGTCAA Aligner 14 10 KS072012 14 AGTTCC Aligner 15 10 KS072012 15 ATGTCA Aligner 16 10 KS072012 16 CCGTCC Aligner 17 10 KS072012 17 GTAGAG Aligner 18 10 KS072012 18 GTCCGC Aligner 19 10 KS072012 19 GTGAAA Aligner 20 10 KS072012 20 GTGGCC Aligner 21 10 KS072012 21 GTTTCG Aligner 22 10 KS072012 22 CGTACG Aligner 23 10 KS072012 23 GAGTGG Aligner 24 10 KS072012 24 GGTAGC F 753 3UMRevD KS073012UD Active Date 11012013 a iochain waw b
3. Water as dilution if necessary in a sterile nuclease free 200 yl PCR tube on ice 2 Add 2 ul Tail Oligo into RNA tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly 3 Incubate the tube at 70 C for 2 minutes and then immediately place the tube on ice 4 Transfer 2 ul Tail Buffer to a sterile nuclease free 200 ul PCR tube on ice F 753 3UMRevD KS073012UD Active Date 11012013 Cd J BioChain 5 Add 2 ul Tail Enzyme to the Buffer tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly 6 Transfer these 4 ul mixture to RNA tube from Step 3 Gently pipette the entire volume up and down 6 8 times to mix thoroughly Incubate the tube at 28 C for 1 hour 7 Directly add 2 ul Ligation Enhancer into reaction tube remaining on the thermal cycler gently pipette the entire volume up and down 6 8 times to mix thoroughly continue incubate the tube at 28 C for 15 minutes and then place the tube on ice 8 Aliquot 2 ul Cap Oligo into a separate nuclease free 200 ul PCR tube incubate at 70 C for 2 minutes and then immediately place the tube on ice 9 Add 2 ul of Cap Enzyme to Cap Oligo tube Gently pipette the entire volume up and down 6 10 11 12 13 14 15 16 1 18 19 8 times to mix thoroughly Transfer these 4 ul of the Cap mixture to the Tail reaction tube from Step 7 Gently pipette the entire volume up and down 6 8 time
4. 3UMRevD KS073012UD Active Date 11012013 d C gt Biocnaine ra whigchaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Figure 1 Workflow Chart of Directional mRNA NGS Library Construction Mm mRNA Fragmentation SS gt i aA mA _ Enzyme Treatment 5 P 7 WAWNANNANNAN Purification Bor a 3 Adaptor Ligation EEEE TORE 5 Adaptor Ligation PIEEO EAEE Reverse Transcription PAAA AA AAAA soonecsonecsoncnao PCR Amplification C o DNA Fragment Enrichment a Pa eS BioChain also provides other tools and services to researchers interested in using NGS technologies Please contact BioChain Technical Support for further details Features e Simple workflow most components are supplied as ready to use master mixtures which reduces setup time and liquid handling steps e Leading level of directionality e Wide dynamic range purified mRNA could be down to 50 ng e Automation capable protocol Applications e Expression of all coding RNAs e Identification of alternative splicing events e Detection of single nucleotide polymorphisms or mutations e Discretion of translocations and fusion transcripts e Discovery of allele specific expression patterns F 753 3UMRevD KS073012UD Active Date 11012013 CJ Biochaine wwwbiochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Description Components in this kit are prepared with pure chemical
5. ing 100 ng of purified mRNA from Adult Normal Lung Tissue Total RNA Cat R1234152 50 processed by BioChain s MagSeq mRNA Purification Kit Cat K2012008 The number of PCR cycles can be adjusted to a maximum of 15 cycles if very low amount of product DNA Fragment Enrichment Recommend use Beckman Coulter Genomics Agencourt AMPure XP Beads for PCR clean up Use 11 ul DNA storage Solution for elution Library Validation F 753 3UMRevD KS073012UD Active Date 11012013 www biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Q iochain www biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Agilent Technologies 2100 Bioanalyzer is highly recommended as quality control system to validate DNA library generated from above 1 Use 1 yl resuspended construct for DNA 1000 chip or High Sensitivity DNA chip 2 Check the size purity and concentration of the sample Figure 2 DNA 1000 Chip Trace of the Final Library from a human lung tissue mRNA Sample FU 50 15 50 100 200 300 400 500 700 1500 bp Related Products RapidSeq High Yield Small RNA Sample Prep Kit Cat KS074012 MagSeq mRNA purification Kit Cat K2012008 Adult Normal Lung Tissue mRNA Cat M1234152 References 1 Wang Z et al Nature Reviews Genetics 2009 10 57 63 2 Ozsolak F et al Nature 2009 461 814 818 3 Labaj PP et al ISMB 2011 27 1383 i391 F 753 3UMRevD KS073012UD Ac
6. iochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com KS072012 IIl Item Amount in kit ul Part No Sequence Aligner 25 10 KS072012 25 ACTGAT Aligner 26 10 KS072012 26 ATGAGC Aligner 27 10 KS072012 27 ATTCCT Aligner 28 10 KS072012 28 CAAAAG Aligner 29 10 KS072012 29 CAACTA Aligner 30 10 KS072012 30 CACCGG Aligner 31 10 KS072012 31 CACGAT Aligner 32 10 KS072012 32 CACTCA Aligner 33 10 KS072012 33 CAGGCG Aligner 34 10 KS072012 34 CATGGC Aligner 35 10 KS072012 35 CATTTT Aligner 36 10 KS072012 36 CCAACA KS072012 IV Item Amount in kit pl Part No Sequence Aligner 37 10 KS072012 37 CGGAAT Aligner 38 10 KS072012 38 CTAGCT Aligner 39 10 KS072012 39 CTATAC Aligner 40 10 KS072012 40 CTCAGA Aligner 41 10 KS072012 41 GACGAC Aligner 42 10 KS072012 42 TAATCG Aligner 43 10 KS072012 43 TACAGC Aligner 44 10 KS072012 44 TATAAT Aligner 45 10 KS072012 45 TCATTC Aligner 46 10 KS072012 46 TCCCGA Aligner 47 10 KS072012 47 TCGAAG Aligner 48 10 KS072012 48 TCGGCA Storage and Stability Upon receipt store all reagents appropriately Avoid repeated freeze thaw cycles The shelf life is 6 months for the kit F 753 3UMRevD KS073012UD Active Date 11012013 a CJ Biocnaine www biochain com Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Protocol Consumables Preparation The kit has all key reagents to run expe
7. riment except the common consumables and instruments Please make sure all materials are available before starting this protocol Table 4 Table 4 List of Consumables and Equipments 0 2 ml 1 5 ml and 2 ml clean nuclease free General lab supplier microcentrifuge tubes Nuclease free Water General lab supplier Ethanol General lab supplier 100 mM EDTA General lab supplier RNA clean kit Zymo Concentrator 5 R1015 or Qiagen MinElute 74204 DNA clean kit Zymo Concentrator 5 D4003 or Beckman Coulter Genomics Agencourt AMPure XP Beads A63880 1 2 200 ul clean nuclease free PCR tubes General lab supplier Magnetic stand General lab supplier Thermo Scientific Thermal cycler General lab supplier General lab supplier Benchtop microcentrifuge General lab supplier 2100 Bioanalyzer Agilent DNA 1000 chip Agilent 5067 1504 High Sensitivity DNA chip optional Agilent 5067 4626 Cautions 1 This product is for Research Use Only 2 Close adherence to the protocol will assure optimal performance and reproducibility 3 Set up reactions in sterile nuclease free tubes on ice 4 Prepare 10 extra mixture when running multiple samples 5 Care should be taken to ensure nuclease free processing 6 Due to the analytical sensitivity of this test extreme care should be taken to avoid the contamination of reagents 7 The assay kit should be used as a system Do not substitute other manufacturer s reagents Dilution red
8. s Buffer 2 is preferable for mRNA from animal sources 3 Incubate the tube at 94 C for 5 minutes and then immediately place the tube on ice Note The fragmentation time sometimes need to be optimized from shorter period since some of the fragile RNAs can be completely degraded or fragmented into very short sizes in long period 4 Immediately add 2 uI 100 mM EDTA to the tube Fragmentation Enzyme Treatment 1 Add 5 ul Fragmentation Enzyme Mix to the fragmentation reaction tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly 2 Incubate the tube at 37 C for 60 minutes and then place the tube on ice Treated mRNA Purification Recommend use Zymo RNA Clean amp Concentrator 5 or Qiagen RNeasy MinElute Cleanup kit according manufacturer s instruction Use 10 ul nuclease free water for elution Pooling Each RapidSeq High Yield Directional mRNA Sample Prep Kit can be used to construct libraries that are compatible with illumina multiplexing While processing samples in parallel incorporate the index at the amplification step following reverse transcription Samples could be pooled immediately prior to DNA fragment enrichment or make pools of samples after that Library Preparation Pre heat the thermal cycler to 70 C and pre heat another thermal cycler to 28 C if available 1 Prepare purified Fragmentation Enzyme treated mRNA sample for total volume at 5 ul use Nuclease free
9. s to construct NGS libraries compatible with Illumina s sequencing platform for subsequent cluster generation using purified mRNA as input Four sets of the kit with different 4 sets of 12 aligners are available respectively Quality Control At least one kit of each lot has been tested for directional mRNA NGS library construction using mRNA isolated from BioChain s Adult Normal Lung Tissue Cat M1234152 and Illumina s NGS instrument Good distributions are observed Components One kit with aligner has 3 boxes listed in below only one aligner box is included in one kit see table 1 3 below Reagents are sufficient for 12 assays The kit without aligner only includes 2 boxes Table 1 Contents List of RapidSeq High Yield Directional mRNA Sample Prep Kit Box 1 of 3 Store at 20 C Cap Color Item Amount in kit Part No Nature Fragmentation Buffer 1 30 ul KS073012 1 Nature Fragmentation Buffer 2 30 ul KS073012 13 Amber Fragmentation Enzyme Mix 70 ul KS073012 2 Orange DNA Storage Solution 160 ul LB3401010 Table 2 Contents List of RapidSeq High Yield Directional mRNA Sample Prep Kit Box 2 of 3 Store at 20 C Cap Color Item Amount in kit Part No Tail Oligo 30 ul KS073012 3 Green Tail Buffer 30 ul KS073012 4 Tail Enzyme 30 ul KS073012 5 Ligation Enhancer 27 ul KS073012 6 Red Cap Oligo 30 ul KS073012 7 Cap Enzyme 30 ul KS073012 8 Yellow RT Oligo
10. s to mix thoroughly Incubate at 28 C for 1 hour and then place the tube on ice Add 4 ul RT Oligo to the whole reaction from previous step Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly Incubate at 70 C for 2 minutes and then immediately place the tube on ice Pre heat the thermal cycler to 50 C Add 4 ul of RT Enzyme Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly Incubate at 50 C for 1 hour and then place the tube on ice In a separate sterile nuclease free 200 ul PCR tube set up PCR mixture as below Mixture yl PCR MasterMix 50 Universal Primer 4 Aligner 4 Nuclease free Water 17 Total 75 For each reaction only one of the 48 Aligners is used during this step Gently pipette the entire volume up and down 6 8 times to mix thoroughly centrifuge briefly then place the tube on ice Transfer this 75 ul mixture to the RT reaction tube from Step 16 Gently pipette the entire volume up and down 6 8 times then centrifuge briefly and place the tube on ice Amplify the tube in the thermal cycler using the following PCR cycling conditions 1 98 C for 30 seconds 2 13 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 15 seconds 3 72 C for 10 minutes 4 hold at 4 C Amplification products may vary based on RNA input amount tissue type and species This process was optimized us
11. tive Date 11012013
12. ucing reaction volumes or other deviation in this protocol may affect the performance of this testing kit 8 Do not mix or combine reagents from kits with different lot numbers 9 Materials are stable until the labeled expiration date when stored and handled as directed Do not use kits beyond their expiration date F 753 3UMRevD KS073012UD Active Date 11012013 d C gt Biocnaine ra whigchaincom Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com RNA Input 1 This protocol has been optimized using purified 100 ng mRNA as input from Adult Normal Lung Tissue Total RNA Cat R1234152 50 processed by BioChain s MagSeq mRNA Purification Kit Cat K2012008 2 For positive control BioChain recommends using MagSeq mRNA Purification Kit Cat K2012008 to purify Adult Normal Lung Tissue Total RNA Cat R1234152 50 3 Messenger RNA populations can vary significantly between different tissue types and species Use of mRNA from other species tissues and purification kits may require further optimization mRNA Fragmentation Pre heat the thermal cycler to 94 C 1 Prepare mRNA sample 50 100 ng in a sterile nuclease free 200 uI PCR tube as total volume at 16 ul 2 Add 2 ul Fragmentation Buffer gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly Note Two Fragmentation Buffers are provided in the kit Buffer 1 has a better fragmentation performance on mRNA of plant source
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