High Yield Small RNA Sample Prep Kit
Contents
1. thermal cycler to 50 C 15 Add 4 ul of RT Enzyme Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly 16 Incubate at 50 C for 1 hour and then place the tube on ice 17 In a separate sterile nuclease free 200 ul PCR tube set up PCR mixture as below PCR MasterMix Nuclease free Water 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 w then place the tube on ice 18 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 19 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 F 753 3UMRevA KS07401 2UA Active Date 09172012 sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Amplification products may vary based on RNA input amount tissue type and species This process was optimized using 100 ng of Adult Lung Tissue mRNA The number of PCR cycles can be adjusted to a maximum of 15 cycles if very low amount of product 20 Run sample on a DNA1000 chip according to the manufacturer s instructions The following figure 2 sh2. sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com User s Manual and Instructions RapidSeq High Yield Small RNA Sample Prep Kit Catalog Number KS074012 KS074012 1 KS074012 II KS074012 III KS074012 IV Introduction Small RNA includes microRNA miRNA ncRNA siRNA snoRNA piRNA rasiRNA and many more It is a large family of regulatory molecules in organisms and plays an important role in development and disease Next Generation Sequencing NGS is a powerful tool to identify and quantitatively analyze the entire population of small RNAs miRNAs are endogenous regulators of gene expression that are encoded in the genomes of animals plants and viruses Mature miRNAs are 18 24 nt single stranded molecules that become incorporated into the RNA induced silencing complex RISC RISC mediates down regulation of gene expression through translational inhibition transcript cleavage or both This manual aims to prepare NGS libraries for subsequent cluster generation using purified small RNA or total RNA which contains small RNA fragments as input The protocol includes steps for adapters ligation reverse transcription PCR amplification and size selection by gel purification to generate a library product compatible with illumina NGS platform Figure 1 The method in this kit ligates adapters directionally to miRNAs based on their nature structure with a 5 phosphate and a 3 hydroxy grou
3. NA as input 2 Purified 1 10 ng small RNA or miRNA from total RNA can also be used as starting material Small RNA populations can vary significantly between different tissue types and species Use of RNA from other species tissues or qualities may require further optimization 3 BioChain recommends using Adult Lung Tissue Total RNA catalog R1234152 50 as a positive control sample for this protocol This product is certified to contain the small RNA fraction Pooling Each RapidSeq High Yield Small RNA Sample Prep Kit can be used to construct libraries that are compatible with Illumina multiplexing with up to 12 samples combined into a single lane While processing samples in parallel incorporate the index at the amplification step following reverse transcription Samples could be pooled immediately prior to gel purification Library Preparation Pre heat the thermal cycler to 70 C and pre heat another thermal cycler to 28 C if available F 753 3UMRevA KS07401 2UA Active Date 09172012 sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com 1 Briefly centrifuge the thawed reagents at 600 xg for 5 seconds and place them on ice 2 Prepare RNA sample for total volume at 5 ul use Nuclease free Water as dilution if necessary in a sterile nuclease free 200 ul PCR tube on ice 3 Add 2 ul Tail Oligo into RNA tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly then cent
4. at KS341 025 Broad Range Total RNA Isolation Kit Cat K1341050 BioChain Total RNA containing miRNAs References 1 Cullum R et al Respirology 2011 16 210 222 2 Shalgi R et al Aging 2009 1 762 770 3 Ach R et al BMC Biotechnology 2008 8 69 F 753 3UM RevA KS07401 2UA Active Date 09172012
5. e with Illumina s sequencing platform for subsequent cluster generation using purified small RNA or total RNA contains small RNA fragments as input Four sets of the kit with different 4 sets of 12 aligners respectively are available Quality Control At least one kit of each lot has been tested for small RNA NGS library construction using BioChain s Adult Normal Lung Tissue Total RNA Cat R1234152 50 and Illumina s NGS instrument Good coverage and low adapter dimmer are observed Components One kit with aligner has 3 boxes listed in below only one aligner box is included in one kit See table 2 4 below Reagents are sufficient for 12 assays The kit without aligner only includes 2 boxes Table 2 Contents List of RapidSeq High Yield Small RNA Sample Prep Kit Box 1 of 3 Cap Color Amount in kit_ PartNo Tail Oligo KS074012 3 Green Tail Buffer KS074012 4 Tail Enzyme KS074012 5 Ligation Enhancer KS074012 6 Cap Oligo KS074012 7 Cap Enzyme KS074012 8 Yellow RT Oligo KS074012 9 RT Enzyme KS074012 10 Blue Universal Primer KS074012 11 PCR MasterMix 700 ul KS074012 12 Amber Gel Cutting Indicator 26 ul L5022100 DS Table 3 Contents List of RapidSeq High Yield Small RNA Sample Prep Kit Box 2 of 3 Item Gel Cutter KS071012 11 Gel Breaker KS071012 12 12 KS071012 13 Gel Filter aa ae DNA Storage Solution 1500 ul x 2 LB3401010 F 753 3UM RevA KS07401 2UA Active Date 09172012 siocha BioC
6. hain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Table 4 Contents List of RapidSeq High Yield Small RNA Sample Prep Kit Box 3 of 3 KS072012 1 Tem Amount in it u PartNo Sequence KS072012 7 C moneri 0 KSova01211 _aactac_ Aligner 12 KS072012 12 CTTGTA 10 0 KS072012 II Hem _ Amountinkit u PartNo Sequence Aligner 21 F 753 3UM RevA KS07401 2UA Active Date 09172012 siocha J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com KS072012 III Amountin Kit ul PartNo Sequence KS072012 25 ACTGAT KS072012 26 ATGAGC Aligner 25 KS072012 27 ATTCCT Miers 0 ksorz201235 _caTTTT K5072012 3 Aligner29 10 KS072012 29 CAACTA F i i l 7 i 7 I l 0 0 0 0 0 0 0 0 0 0 0 0 KS072012 IV amount in kitu PartNo Sequence Aigner 40 Aligner 4 Aligner 42 KS072012 42 70 0 Aligner 37 Aligner 38 Aligner 39 Aligner 43 KS072012 43 TACAGCG Aligner 44 KS072012 44 TATAAT Aligner 45 KS072012 45 TCATTC Aligner 46 KS072012 46 TCCCGA Aligner47 10 KS072012 47 TCGAAG Aligner 48 KS072012 48 TCGGCA Storage and Stability Upon receipt store all reagents appropriately Avoid repeated freeze thaw cycles This kit is stable for half a year after shipping date F 753 3UM RevA KS074012UA Active Date 09172012 sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Ema
7. il info biochain com Protocol Consumables Preparation The kit has all key reagents to run experiment but not common consumables and instruments Please make sure all needs are available before starting this protocol Table 5 Table 5 List of Consumables Consumable 0 2 ml 1 5 ml and 2 ml clean nuclease free General lab supplier microcentrifuge tubes 200 ul clean nuclease free PCR tubes General lab supplier 5X Novex Hi Density TBE Sample Buffer Invitrogen LC6678 Cautions This product is for Research Use Only Close adherence to the protocol will assure optimal performance and reproducibility Set up reactions in sterile nuclease free tubes on ice Prepare 10 extra mixture when running multiple samples Care should be taken to ensure nuclease free processing 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 reducing 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 OnaRWN RNA Input 1 This protocol has been optimized using 1 yg of high quality human lung total R
8. iochain com ProtocolManuals Gel_Purification_Manual pdf 12 Place the band of interest into the 0 5 ml Gel Breaker tube 13 Centrifuge the stacked tubes to 20 000 g in a microcentrifuge for 2 minutes at room temperature Ensure that all the gel has moved through the holes into the bottom tube 14 Remove Gel Breaker tube add up to 200 ul of DNA Storage Solution to the gel debris in the 2 ml tube F 753 3UMRevA KS07401 2UA Active Date 09172012 siocha J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com 15 Elute the DNA by shaking the tube around 1300 rpm at room temperature for at least 2 hours or overnight if desired 16 Transfer the eluate and the gel debris to the top of a 5 um filter 17 Centrifuge the filter for 10 seconds to 600 g and then discard the filter 18 Check the size purity and concentration of the library on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip Figure 4 Figure 4 High Sensitivity DNA Chip Trace of the Final Library from an Adult Normal Lung Tissue Total RNA Sample Peak 1 Lower Marker Peak 2 miRNA NGS Library Peak 3 Upper Marker Related Products RapidSeq MasterMix Small RNA Sample Prep Kit Cat KS071 012 RapidSeq MasterMix Directional mRNA Sample Prep Kit Cat KS073012 RapidSeq High Yield Directional mRNA Sample Prep Kit Cat KS075012 MagSeq mRNA Purification Kit Cat K2012008 MicroRNA Isolation Kit C
9. ows typical result from Adult Normal Lung Tissue Total RNA Figure 2 Adult Normal Lung Tissue Total RNA Sample Trace of Amplicons on DNA1000 Chip Heq yyw Size Selection by Gel Purification 1 Assemble the gel electrophoresis apparatus per the manufacturer s instructions with appropriate amount of 1X TBE Running Buffer 2 Mix 2 ul of Gel Cutting Indicator with 2 ul of DNA Loading Buffer 5X Novex Hi Density TBE Sample Buffer or equivalent 3 Load 2 ul per lane of Gel Cutting Indicator in outer side of sample wells 4 Load maximum 30 ul cDNA library with appropriate amount of DNA Loading Buffer each well 5 Run the 6 TBE gel for 60 minutes or 8 TBE gel for 65 minutes to get better separation at 145 V or until the blue front dye exits the gel 6 Remove and stain the gel with Ethidium Bromide 0 5 ug ml in water for 2 3 minutes 7 View the gel on a Dark Reader transilluminator or a UV transilluminator Figure 3 Small RNA Library from an Adult Normal Lung Tissue Total RNA Sample Lane 1 and 2 Gel Cutting Indicator for miRNA NGS library size selection Lane 3 and 4 Amplicons of an Adult Normal Lung Tissue Total RNA Samples 10 Place the gel breaker tube into a sterile round bottom nuclease free 2 ml microcentrifuge tube 11 Using a Gel Cutter cut out miRNA NGS library band between two Cutting Indicators and excise the gel fragment Please refer to Gel Cutter Users Manual online version http www b
10. p F 753 3UMRevA KS07401 2UA Active Date 09172012 sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Figure 1 Workflow Chart of Small RNA NGS Library Construction Purified Small RNA or Total RNA Contains Small RNA Fragments 3 Adapter Ligation 5 Adapter Ligation First Strand Synthesis Reverse Transcription Amplification with Index PCR Gel Purification BioChain also provides other tools and services to researchers interested in studying small RNA Please contact BioChain Technical Support for further details Features e Simple workflow most components are supplied as ready to use super mixtures which reduces setup time and liquid handling steps Table 1 e Great performance comparable yield with benchmark s fresh made mixtures e Wide dynamic range total RNA input could be down to 100 ng Table 1 Savings in manual time and effort with BioChain method Total process time 3 Adapter Ligation 1 hr 15 min 5 Adapter Ligation eon om om Applications e Small RNA detection and quantification e Small RNA discovery e MicroRNA expression profiling e MicroRNA related functional assessment and validation F 753 3UMRevA KS07401 2UA Active Date 09172012 sioca J BioChain Tel 1 888 762 2568 Fax 1 510 783 5386 Email info biochain com Description Components in this kit are prepared with pure chemicals to construct NGS libraries compatibl
11. rifuge briefly 4 Incubate the tube at 70 C for 2 minutes and then immediately place the tube on ice 5 Transfer 2 ul Tail Buffer to a sterile nuclease free 200 ul PCR tube on ice 6 Add 2 ul Tail Enzyme to the Buffer tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly 7 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 8 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 9 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 10 Add 2 ul of Cap Enzyme to Cap Oligo tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly 11 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 times to mix thoroughly 12 Incubate at 28 C for 1 hour and then place the tube on ice 13 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 14 Incubate at 70 C for 2 minutes and then immediately place the tube on ice Pre heat the
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