User`s Manual
Contents
1. RapidSeq Small RNA Sample Prep Kit 1011V2 User s Manual and Instructions Product RapidSeq Small RNA Sample Prep Kit Catalog Number KS071012 KS071012 I KSO71012 II KSO71012 III and KS071012 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 hydroxyl group Figure 1 Workflow Chart of Small RNA NGS Libra2. Sample Prep Kit Box3 of 3 KS07 1012 1 tem Amountin kit u PartNo Sequence KS072010 4 K5072012 6 KS072010 9 KS07101241 Amount in kt u PartNo Sequence Aligner 15 Aligner 16 Aligner 17 Aligner 18 KS072012 18 1 Aligner 13 Aligner 14 Aigner 19 Aligner 20 Aigner 21 Aigner 22 Aligner23 10 _x072012 29 GAGTGG Aligner 24 KS072012 24 GGTAGC 0 0 0 0 0 0 0 0 0 0 0 0 Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 KS07 1012 111 TAmountin kit ul PartNo Sequence Aligner 25 Aligner 26 Aligner 27 KS072012 27 Aligner 28 Aligner 29 Aligner 30 Aligner 3 Aligner 32 Aligner 33 Aligner 34 Aligner 35 KS072012 35 Aligner 36 KS072012 36 CCAACA KS071012 IV Amount inkt PartNo Sequence Aigner 39 Aigner 40 Aigner 4 Aigner 42 1 Aligner 37 Aligner 38 Aligner 43 Aigner 44 Aligner 45 Aligner 46 KS072012 46 Aigner47_ 0 ksorzo247 TCGAAG Aligner 48 KS072012 48 TCGGCA Storage and Stability 0 0 0 0 0 0 0 0 0 0 0 0 Upon receipt store all reagents appropriately Avoid repeated freeze thaw cycles This kit is stable for half a year after shipping date Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 Protocol C
3. al Lung Tissue Total RNA Sample Lane 1 and 2 Gel Cutting Indicator for miRNA NGS library size selection Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 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 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 the gel has all moved through the holes into the bottom tube 14 Remove Gel Breaker tube add 200 ul of DNA Storage Solution to the gel debris in the 2 ml tube 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 Low
4. d 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 15 Pre heat the thermal cycler to 50 C 16 Add 5 5 ul of RT MasterMix Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly 17 Incubate at 50 C for 1 hour and then place the tube on ice 18 In a separate sterile nuclease free 200 ul PCR tube set up PCR mixture as below NO PCR MasterMix Universal Primer 2 Aligner 2 Nuclease free Water Total 7 5 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 19 Transfer this 37 5 ul mixture to the RT reaction tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly and place the tube on ice 20 Amplify the tube in the thermal cycler using the following PCR cycling conditions Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 1 98 C for 30 seconds 2 11 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 15 seconds 72 C for 10 minutes 3 Hold at 4 Amplification products may vary based on RNA input amount tissue type and species This process was optimized using 1 yg of A
5. dult Lung Tissue Total RNA The number of PCR cycles can be adjusted to a maximum of 15 cycles if no clear bands in the gel image 21 Run sample on a DNA1000 chip according to the manufacturer s instructions The following figure 2 shows typical results from Adult Normal Lung Tissue Total RNA Cat R1234152 50 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 Mix 2 ul of Gel Cutting Indicator with 2 ul of DNA Loading Buffer 5X Novex Hi Density TBE Sample Buffer or equivalent Mix amplified cDNA library with appropriate amount of DNA Loading Buffer Load 2 ul per lane of Gel Cutting Indicator in outer side of sample wells Load maximum 30 ul cDNA library each well in between two Indicator wells Run the gel for 60 minutes at 145 V or until the blue front dye exits the gel Remove the gel from the apparatus and open the cassette according to the manufacturer s instructions 8 Stain the gel with Ethidium Bromide 0 5 ug ml in water in a clean container for 2 3 minutes 9 View the gel on a Dark Reader transilluminator or a UV transilluminator N NOONA The following figure 3 shows gel analysis of an Adult Normal Lung Tissue small RNA library Figure 3 Small RNA Library from an Adult Norm
6. eled expiration date when stored and handled as directed Do not use kits beyond their expiration date RNA Input 1 This protocol has been optimized using 1 yg of high quality human lung total RNA as input Use of degraded RNA can result in low yield 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 BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 Pooling Each RapidSeq 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 1 Briefly centrifuge the thawed reagents at 600 xg for 5 seconds then place them on ice 2 Prepare RNA sample
7. er Marker Peak 2 miRNA NGS Library Peak 3 Upper Marker Related Products MagSeq mRNA Purification Kit Cat K2012008 MicroRNA Isolation Kit Cat KS 341025 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 BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com
8. for total volume at 5 ul use Nuclease free Water as dilution if necessary in a sterile nuclease free 200 pl PCR tube on ice 3 Add 1 ul Tail Oligo into RNA tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly then centrifuge briefly 4 Incubate the tube at 70 C for 2 minutes and then immediately place the tube on ice 5 Add 4 ul of Tail MasterMix to the reaction tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly Incubate the tube at 28 C for 1 hour Directly add 1ul Ligation Enhancer into each 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 1 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 MasterMix to Cap Oligo tube for each reaction Gently pipette the entire volume up and down 6 8 times to mix thoroughly 10 Transfer these 3 pl of the Cap mixture to the Tail reaction tube Gently pipette the entire volume up and down 6 8 times to mix thoroughly 11 Incubate at 28 C for 1 hour and then place the tube on ice 12 Aliquot 6 ul of the whole reaction into a separate sterile nuclease free 200 ul PCR tube Left could be stored at 80 C 13 Add 1 ul RT Oligo Gently pipette the entire volume up an
9. ina s sequencing platform for subsequent cluster generation using purified small RNA or total RNA contains small RNA fragments as input 4 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 dimer are observed All known miRNAs are captured Components One kit 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 Table 2 Contents List of RapidSeq Small RNA Sample Prep Kit Box 1 of 3 Cap Color hem Amountin kit_ PartNo 14 pi KS071012 1 Ligation Enhancer 14ul KS071012 3 Tail Oligo Green Tail MasterMix 53 ul KS071012 2 Cap MasterMix 26 5 ul KS071012 5 Cap Oligo 14 ul KS071012 4 Yelow D a a A Blue Gel Cutting Indicator 26 ul L5022100 DS BioChain Institute Inc t 888 762 2568 f 510 783 5386 Website www biochain com e mail info biochain com RapidSeq Small RNA Sample Prep Kit 1011V2 Table 3 Contents List of RapidSeq Small RNA Sample Prep Kit Box 2 of 3 Item Amountinkit_ PartNo KS071012 11 Gel Breaker KS071012 12 Gel Filter KS071012 13 DNA Storage Solution 1500 ul x 2 LB3401010 Table 4 Contents List of RapidSeq Small RNA
10. onsumables 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 5X Novex TBE Buffer Invitrogen LC6675 6 Novex TBE PAGE Gel 1 0 mm 10 well Invitrogen EC6265BOX DNA 1000 chip Agilent 5067 1504 Ultra Pure Ethidium Bromide General lab supplier High Sensitivity DNA chip 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 manufacturers 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 lab
11. ry Construction Purified Small RNA or Total RNA Contains Small RNA Fragments 3 Ada pte rLi ga tion VAARNAA 5 Ada pte Eii ga tion MAA ARAL 8979 First Strand Synthesis Manan pooocscocacacacaogo Reverse Transcription Amplification with Index PCR O 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 BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com RapidSeq Small RNA Sample Prep Kit Table 1 Savings in manual time and effort with BioChain method Total process time 1 hr 15 min Protocol Hands on time 3 Adapter Ligation 5 Adapter Ligation First Strand Synthesis C Size selection Purification Total workflow time Applications e Small RNA detection and quantification e Small RNA discovery e MIRNA expression profiling e MiRNA related functional assessment and validation Description 1011V2 Components in this kit are prepared with pure chemicals to construct NGS libraries compatible with Illum
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