LncProfiler™ qPCR Array Kit
Contents
1. Did you select SYBR Green as the Detector s Reporter Dye Did the controls work Use more cDNA in Mastermix Check Mastermix contents and try a subset with the controls as a positive control Also try lowering the Annealing Temperature to 55 C How do select the Threshold level Typically place the threshold setting in for Ct analysis the center of the exponential phase of the amplification curve Also see the User Manual for your specific instrument or phone their technical support team for guidance 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual V LncRNA Technical References selected Wang KC Chang HY Molecular Mechanisms of Long Noncoding RNAs Mol Cell 2011 Sep 16 43 6 904 14 Sotillo E Thomas Tikhonenko A The long reach of noncoding RNAs Nat Genet 2011 Jun 28 43 7 616 7 doi 10 1038 ng 870 Guttman M J Donaghey B W Carey M Garber J K Grenier G Munson G Young A B Lucas et al 2011 lincRNAs act in the circuitry controlling pluripotency and differentiation Nature 2011 Aug 28 doi 10 1038 nature10398 Cabili M N C Trapnell L Goff M Koziol B Tazon Vega A Regev and J L Rinn 2011 Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses Genes Dev 2011 Sep 2 Cunnington MS Santibanez Koref M Mayosi BM Burn J Keavney B Chromosome 9p21 SNPs Associated with Multiple Dis2. Follow the guidelines as detailed for your specific Real time instrumentation The following parameters tested by SBI were performed on an Applied Biosystems 7300 7500 Real time PCR System but can also apply to any other 96 well systems The details of the thermal cycling conditions used in testing at SBI are below A screenshot from the Real time instrument setup is shown below also Default conditions are used throughout 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 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Instrument setup qPCR cycling and nanasa data accumulation d Pam cpr noca conditions A humans oes mai Profle Jato increment Ramp Asie Dais Cofector Tha 1 Shae F ages Standard Protocol Raves 3 1 50 C 2 min 2 95 C 10 min 3 95 C 15 sec 4 60 C 1 min 40 cycles of Stage data read at 0 C 1 min Step 3 6 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 thres
3. sens m a Be T ees gt LncRNAs e D 2 ia Te i of j System Biosciences a3 LncProfiler qPCR Array Kit Quantitate long non coding RNAs IncRNAs by real time qPCR Cat RAQ00A 1 RA910A 1 User Manual Store kit at 20 C on 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 LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 Contents I Introduction and Background A Overview ss 2 B LncProfiler qPCR profiler workflow s s sSseseses 5 C How LncProfiler cDNA synthesis works 6 D Listofcomponents ss T7 ll Protocol A LncProfilercDNAreactionsetup ____ sss s s s sss 8 B Real time qPCR Reaction setup 2 9 C How the IncRNA specific primers are designed _ _s_______ 10 D LncProfiler qPCR array contents 0 12 E Endogenous reference controls 2 13 lll Sample Data and Quality Control A LncProfiler qPCR sample data 2 14 B Specificity Tests ss i i i i i iti 16 IV Troubleshooting 17 V LncRNA Selected References sss lt lt 18 VI Technical Support C 18 IX Licensing and Warranty Statement 19 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction and Background A Ove
4. D200 Bl and G22 beet form a faery of mlepercevily exapted repetilha eierments which have eyobeed bo Carty oul ila funtion in dierent manman species Mucikigsmatty 2002 Chana LETT More than 200 priores pd utes N Daa Genoa withe NCHA dipnasian CMG SOANA anges 11 human ties uing palaistra amplicahion Trarclational control by a smal ANd dendritic BTI RHA targets the eubaryobe initiation factor 44 helaa mechanion Tep primab soecifie and non oroben coding RMAs in bregar iot neuronal Gepdesion publ localization ara benches patres 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual D LncProfiler qPCR array contents The qPCR array plate contains assays for 90 IncRNAs and also includes 5 endogenous reference RNAs as normalization signals Please see the SBI website to download the qPCR array arrangement and AACT analysis software www systembio com LncRNA 3 4 6 7 8 9 11 12 Evf1 and H19 upstream _matarr masma mecs meco merne navuran woma meani mesas meon nr ps3 mRNA PCGEM1 i PTENP1 SCA8 inhibiting jee il i Sa SES ra Ta r a as EL S3124 855 ERINA EN paa 112679 947 112578247 ODE SS 12 ThbRSE 012 ti ai ean 100 roan 213 AL a ata d o Oa o n s E jj Paste raw Ct Paste raw Ct Data for Test Data for Control Sample in Sample in Column C Column H Your Data is analyzed automatically with geometric mean Normalization The Fold c
5. O07 Transcribed by RMA polymerase Mi Mortiqnett Charechorstics usiz Thhes sinatura domane S that fangs homakagy with Alu Slementt 2 Central A moh rapan and a a umeque ragan Dados 19731 Expressed predominanti in different regars of the bran Also shows lew level expression in tests but pot in other normal tissues geamined haten 1507 Teda 1900 harhar A001 ANA sequencing of 11 human teues confirmed upregulation of expression in brain hypothalamus and low of no eapression etseevhere Carte 201th Dienagquittied im cancer exqnnested ina numer af human tumort but mot in comecpanding norma Goa Chen 1 oo Link with aging and Ahri chara BEAD Gprissaon ienaa i ogre but S Leonia in Ahr eau AD bn AD hecbed bran nagar piir Fora with dee aiit RHA iocakoon shored inher pokey Varn shoeing budd uo of PAA ii oul badr Mie 00T Lite BCI ncRALA found in rodents DC200 aso suggested to ocase to dendrites Tety 9332 Back Gira ponies reag thee agra cognition paricky SAG 14 hebi eoan LFA ryote initiation factor 14 Fico FA fli S008 ond Poh LA nding poba PABP nding fo FASE mom Cintra A ieh gr uhri nee Inhibits mandation in wing and in cultured cels andar to BCL B200 binding to eIF4A inivkats R by uncoupling alF4A ATPase activity fom its helzasa dupes unwinding acteety un 2000 Tharslabional inhibition aso involves B200 binding to to PABA ioni atgw 20051 Anthro ta morkoins aot and puma Sa 1958
6. info systembio com Technical Support tech systembio com Ordering Information orders systembio com Page 18 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 IX Licensing and Warranty Statement Limited Use License Use of the LncProfilers 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 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 SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will repla
7. Ri Dio3 0s family pommi DLG2AS family HARIB memme Malati i MERIC meem HOTAIRM Hoxal la HOXA6as IGF2AS Pn p lincRNA SFMBT2 Loc285194 MEG3 family E H7 hESC mhiPSC Normalized Expression Protocol per one well of 6 well plate Confluent cells in a 6 well remove media Add 1ml Trizol directly to cells on plate Incubate at Room for 5 minutes for complete lysis Collect Trizol cell mixture and transfer to 1 5ml tube Add 200 ul Chloroform vortex 15 seconds Centrifuge mixture for 15 minutes at 4 C Collect aqueous layer and transfer to fresh 1 5 ml tube Add equal volume 250 ul Isopropanol mix by inversion CONOORWND Page 14 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 9 Precipitate RNA overnight at 20 C 10 Centrifuge at 13 000 rom for 20 minutes 11 Remove supernatant 12 Wash 1X with 500 ul 80 Ethanol 13 Centrifuge again for 5 minutes at 13 000 rom 14 Remove supernatant and let air dry 5 minutes 15 Resuspend RNA pellet in 50 ul water RNase free 16 Use 5 ul of RNA per cDNA synthesis LncRNaAs are present in serum exosomes There is high interest in discovering and developing useful RNA based biofluid markers The RNAs in patient fluids are present 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 f
8. model systems SBI has built a sensitive accurate and robust qPCR array to enable researchers to closely profile the expression changes in the top IncRNAs known to date Page 2 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 This manual provides details and information necessary to use the LncProfiler Kit to tag and convert small non coding RNAs into detectable and quantifiable cDNAs The system allows for the ability to quantitate dynamic fold differences of IncRNAs across 20 separate experimental RNA samples The array plate also includes 5 endogenous RNA assays as normalization signals To ensure optimal results please read the entire manual before using the reagents and material supplied with this kit These LncProfiler qPCR Array comes with all the reagents necessary to tag and long non coding as well as small RNAs from 20 different total RNA samples into quantifiable cDNA The kits include assays in pre formatted plates for well annotated human IncRNAs with three endogenous reference RNA controls on each plate All of the IncRNAs on the qPCR array have validated primer sets for well annotated IncRNAs that are registered in the IncRNA database created by Dr John Mattick www Incrnadb org 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Potential functions of IncRNAs To date IncRNAs have been found to exhibit a wide range of funct
9. NAs have been found to participate in imprinting processes Imprinting Control Regions ICRs are DNA regions that are differentially methylated depending on their parental origins Unmethylated ICRs cause specific expression of nearby IncRNAs which then suppress neighboring genes in cis Airn and Kcnqioti are examples of IncRNAs that Cause suppression of paternally inherited genes Gene dosage and X chromosome inactivation The discovery of Xist was one of the defining moments in the realization that ncRNAs can have profound roles in the control of gene expression Xist is an IncRNA that suppresses the inactive non coding X chromosome Xi in female cells In all 7 ncRNAs are found as part of the X inactivation center on the X chromosome including Xist Initially Xist and its antisense transcript Tsix are expressed on both X chromosomes However Tsix expression continues on the X that will remain active Xa and this activity recruits DNMT3A to suppress Xist from being transcribed on Xa On Xi it is Tsix that is suppressed potentially via another IncRNA that is part of the X inactivation center Jox With Tsix suppressed the protein PRC2 is recruited to induce histone modification marks at the 5 end of Xist This upregulates Xist expression on Xi and causes further propagation of these silencing marks throughout Xi which are maintained across the lifetime of the organism Embryonic develooment_and segmentation The expression of HOX genes is al
10. adenylation step greatly enhances cDNA synthesis yields of IncRNAs over 100 fold and enables the usage of small RNAs like U6 and RNU43 to be included as reference controls on the qPCR array aasi om Pu LncProfiler cDNA polyA random primers Normalized ie Signals 558z Random primers PolyA Random primers Page 6 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 D List of components Oligo dT Adaptor 20 ul RT Reaction 5X Reverse Transcriptase Buffer Random Reverse Transcriptase 0 1 M Dithiothreitol DTT 4 2 mi RNase free Water nm ht ea f ACTIONS iw wu ALEE Catalog RA910A 1 contains all of the components listed above Catalog RA900A 1 has all of the above components except for the 2X SYBR Green reagent The kit is shipped on blue ice and should be stored at 20 C upon arrival Properly stored kits are stable for 1 year from the date received The oligonucleotides for the specific IncRNAs are dried down in the wells of the optical qPCR plates Resuspend in 10ul RNase free water SBI recommends using the LncProfiler qPCR array with the following SYBR Green reagents e 2X Maxima SYBR Green with Rox Cat K0223 from Fermentas e Power SYBR Master Mix Cat s 4868577 4367650 4367659 4368706 4368702 4368708 4367660 from Applied Biosystems 888 266 5066 Toll Free 650 968 2200 outside US Page 7 Syst
11. ce 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 888 266 5066 Toll Free 650 968 2200 outside US Page 19
12. ciation analyses for reference controls and some IncRNA qPCR assays performed in duplicate HOTAIR KCNq tot IneRNA IncRNA Bc200 IheRNA Tm 83 6 C Tm 78 9 C f i a Hee Ei A 14 hy Inc AMA A Tm 75 8 C HinHH i jl i j y NN GAPDH i Lamin A c i 185 rRNA Tm 79 C Tm 77C Clean assay design with no background Total RNA was prepared from human HT1080 cells in culture As a control 2ug of this RNA was checked in a mock cDNA synthesis reaction where the reverse transcriptase RT was left out The sample was then tested across the LncRNA Profiler GPCR assays Separately we spiked in 10ng of human genomic DNA and tested this sample with the qPCR assays as well There is ZERO background in the minus RT controls and the LncRNA Profiler assays do not show any amplification signals even with spiked in genomic DNA Profile with confidence and only detect IncRNAs Zero background Assays do not in minus RT Controls _ detect Genomic DNA Gel analysis Sample gel analyses for selected IncRNA qPCR assay amplicon products from HFF cDNA and separated on a 1 5 agarose gel and stained with ethidium bromide Page 16 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 IV Troubleshooting Problem Possible Solution Too much background in qPCR Use much less cDNA in the SYBR signals Green Mastermix
13. ease Phenotypes Correlate with ANRIL Expression PLoS Genet 2010 Apr 8 6 4 e1000899 Kogo R Shimamura T Mimori K Kawahara K Imoto S Sudo T Tanaka F Shibata K Suzuki A Komune S Miyano S Mori M Long non coding RNA HOTAIR regulates Polycomb dependent chromatin modification and is associated with poor prognosis in colorectal cancers Cancer Res 2011 Aug 23 Bellucci M Agostini F Masin M Tartaglia GG Predicting protein associations with long noncoding RNAs Nat Methods 2011 Jun 8 6 444 5 Perez DS Hoage TR Pritchett JR Ducharme Smith AL Halling ML Ganapathiraju SC Streng PS Smith DI Long abundantly expressed non coding transcripts are altered in cancer Hum Mol Genet 2008 Mar 1 17 5 642 55 Mus E Hof PR Tiedge H Dendritic BC200 RNA in aging and in Alzheimer s disease Proc Natl Acad Sci U S A 2007 Jun 19 104 25 10679 84 Peterlin BM Brogie JE Price DH 7SK snRNA a noncoding RNA that plays a major role in regulating eukaryotic transcription Wiley Interdiscip Rev RNA 2011 Aug 18 doi 10 1002 wrna 106 VIII 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
14. em Biosciences SBI User Manual Il Protocol A LncProfiler cDNA reaction setup for 1 RNA sample to be assayed on qPCR 96 well plates the IncRNA fraction RNA input can be as low as 1 2 ug total For optimum signals perform the following 7 It is important to start with total RNA that includes ED Dilute your RNA to 200 400 ng ul Start i In a thin walled PCR tube or s PCR compatible plate well combine 5 ul Total RNA 1 2 ug STEP 1 2 ul 5X PolyA Buffer 1 pl 25mM MnCl PolyA Tail 1 5 ul 5mM ATP 0 5 ul PolyA Polymerase 10 ul Incubate for 30 min at 37 C Add 0 5 ul Oligo dT Adapter STE P 2 z Heat for Ya at 60 C Anneal Anchor Let cool to room temp for 2 min dT Adapter Add 4 ul 5X RT Buffer 2 ul ANTP mix STEP 3 1 5 u 0 1M DTT Synthesize 1 5 pl Random Primer mix cDNAs 1 ul Reverse Transcriptase 20 5 ul Total in tube l Incubate for 60 min at 42 C Heat for 10 min at 95 C The IncRNA cDNAs can be stored at 20 C For more sensitive applications a single D O n l phenol chloroform extraction with ethanol precipitation can be performed on the cDNA to z remove proteins unused dNTPs and primers typically this is not necessary Page 8 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 B Mastermix qPCR Reaction Setup for 1 entire 96 well qPCR plate To determine the expression profile for the IncRNAs under study mix the f
15. ether may impact transcriptional activation or repression Some examples of scaffold IncRNAs are HOTAIR and ANRIL l Signaling ll Guides Promote chromatin IncRNA modification IncRNA Gene Activation l SAA WIA SANI lll Decoy IV Scaffolds Act on chromatin structure IncRNA Q Gene Suppression Page 4 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 B LncProfiler qPCR profiler workflow Control Sample Test Sample Total RNA dl v LncRNA Tail and Tag cDNA synthesis ie Combine LncRNA cDNA 2X SYBR Green Pipet mastermix ry into 96 well plates Add 2 ul of each IncRNA assay eachwell W Se Perform real time qPCR runs Cross compare AACt measurements between Control and Tumor Samples 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual C How the LncProfiler cDNA synthesis works 10 pg 10 pg LncRNAs total RNA anchor tailed IncRNAs 5 3 polyA gt Tag LncRNAs polymerase 4 polyA tailed 5mm AAAAAAAAA 3 IncRNAs Incubate at 37 C 30 min Anneal Adaptor 5 ees AAAAAAAAA 3 2 P NVTTTTTTTTT S 5 Anneal adaptor 60 C 5 min 5 m AAAAAAAAA 3 Convert to cDNA 3 lt TTTTTTTTT eS RT random primers to make cDNA 42 C 60 min G cDNA pool of anchor tailed IncRNAs Ss TTTTTTTTT A 5 cDNA ready for LncProfiler qPCR array The initial poly
16. ffers over 10 log fold of expression range detection 100000000 10000000 m 1080 cells 1000000 4 io0000 M 293 cells rocco E H7 ESCs 00 E hiPSCs hh To explore the IncRNA expression pattern differences between H7 stem cells and iPSCs we next profiled the source cells that were used to make the iPSCs HFF cells The LncProfiler GPCR Array was tested using 2 ug total RNA extracted from H7 human embryonic stem cells H7 hESC human induced pluripotent stem cells hiPSC and human foreskin fibroblast cells HFF The sample RNAs were converted to cDNA LncProfiler Kit The resulting cDNAs were tested using about 10 ng cDNA per well Shown below are the resulting Real time amplification plots for selected data The Ct data were normalized and shown as Normalized Expression levels in the Bar Graph below for some selected data The H7 and iPS cells profile was similar as expected The iPS source cell s HFF IncRNA expression pattern was very different from the stem cells especially for Anril HariA Car Intergenic 10 and Malatt ad o 0 001 0 0001 0 00001 a 0 000001 EFEFEF Normalized Expression Levels 2A eee goA poum Emx205 Meee OCS Ay meee snak oe ee ee eee SNHG3 NTT p a PCGEM 1 H19 pom H19 Upst 14 m SAF 75L AK0O23948 pommi PRINS PTENP 1 0 OOOOH Nospas mmmmmpmmmm oe Alpha 250 p anti NOS2A memmy O BACEIAS family mmmn CAR intergenic 10
17. hange levels are in Column M Page 12 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 Five Subcellular reference controls LncRNAs can localize and function in the nucleolus nucleus and in the cytoplasm The LncProfiler qPCR array includes RNA reference controls to allow for subcellular fractionation studies to identify and profile three separate subcellular compartments Nucleolus SnoRNA RNU43 and some 18S rRNA Nucleus Small Nuclear splicing snRNA U6B Cytoplasm GAPDH Lamin A C and 18S rRNA y Nucleolus Nucleus _ F al Profile your IncRNAs wherever they are located within the cell Example of reference control amplification plots Results may vary depending upon the cell types analyzed 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual lll Sample Data A LncProfiler qPCR Array sample data The LncProfiler qPCR arrays was tested across Human HT1080 lung cancer cells HEK293 embryonic kidney cells H7 human embryonic stem cells and Human induced Pluripotent stem cells hiPSCs The hiPSCs were made using standard Yamanaka retroviruses for Oct4 Sox2 KIf4 and c Myc Intriguingly there are similar but not identical IncRNA expression patterns between H7 stem cells and hiPSC cells The IncRNA expression patterns differ significantly between HEK293 cells and HT1080 lung cancer cells The LncProfiler qPCR array o
18. holds should be set within the exponential phase of the amplification plots with software automatic baseline settings C How the IncRNA specific primers are designed for detection and quantitation in the qPCR array SBI s LncProfiler is complete cDNA synthesis kit combined with a 96 well based qPCR assay set The qPCR assays have been validated across numerous cell types for robust and specific performance Some IncRNAs have endogenouse polyA tails while other IncRNAs do not To enhance qPCR assay performance the cDNA synthesis kit includes reagents to polyadenylate all IncRNAs before cDNA conversion with the oligo dT adaptor and random primers SBI s IncRNA qPCR assays are derived from published primer sequences and others were designed in house for robust performance and all amplicons are below 200 bp in size All qPCR assays are designed to detect Human IncRNAs that are annotated in Dr John Mattick s LncRNA database http www incrnadb org Below is a screenshot of the IncRNA database interface the example is for the bc200 IncRNA database entry where you can find useful information about its discovery expression function conservation as well as some citations for the IncRNA Page 10 ver 1 091911 www systembio com LncProfiler Long Non coding RNA qPCR ArraysCat RA900A 1 RA910A 1 Incma db be200 aliases BCVRA brain cytoplasmic RALA annotation 200 muciestide mcf Tiedee 1993 axamted from an Alu element gteom
19. ions ranging from signaling serving as molecular decoys guiding ribonulceoprotein complexes to specific chromatin sites and also participating as scaffolds in the formation of complexes I Signaling The transcription of certain IncRNAs is very tissue and temporal specific Their expression can be in response to certain stimuli such as cellular stress and temperature Thus IncRNAs can serve as molecular signals and can act as markers of functionally significant biological events Examples include imprinting IncRNAs XIST AIR and Kenqiot1 ll Decoys The molecular decoy type of activity takes place when specific IncRNAs are transcribed and then bind to and titrate away protein factors Decoy IncRNAs can sponge protein factors such as transcription factors and chromatin modifiers This leads to broad changes in the cell s transcriptome Example is MALAT1 lll Guides LncRNAs can be molecular guides by localizing particular ribonucleoprotein complexes to specific chromatin targets This activity can cause changes in gene expression either in cis on neighboring genes or in trans distantly located genes that cannot be easily predicted by just the IncRNA sequence itself Some example IncRNAs that act as guides are XIST and HOTTIP IV Scaffolds Assembly of complex protein complexes can be supported by IncRNAs linking factors to together to form new functions Some IncRNAs possesses different domains that bind distinct protein factors that altog
20. luid malignant ascite fluids and contain distinct subsets of microRNAs depending upon the tumor from which they are secreted We wanted to test whether IncRNAs may be present in circulation exosomes as well by using the LncProfiler qPCR array We precipitated exosomes from a human pooled serum sample 1ml using SBI s ExoQuick exosome precipitation reagent cat EXOQ5A 1 The exosome vesicles were then lysed and the exosomal RNA purified using SBI s SeraMir kit cat RA806A 1 The resulting exoRNA was converted to cDNA using the LncProfiler cDNA synthesis kit The cDNA was tested in duplicate across all of the LncProfiler qPCR array assay set Purifying exosome RNAs and profiling IncRNAs Serum Simple one step Phenol free b precipitation ee Exosome Lysis k AG Lysate Supernatant BIND 3 minute J y WASH exoRNA lean up pork rr EUNE ai te Exosome SAN ll T pellet SBT Profile cDNA in duplicate across the LncProfiler qPCR array m a Discover new biofluid IncRNA biomarkers using the LncProfiler qPCR array kit Amplification plot of serum IncRNAs Dissociation plots of serum IncRNAs MEER T ee anti PEG EJF 4 aniserne SPMET2 1000 Relative abundance antiPegll E2F4 SFMBT2 SAF YRNA 1 SNHG6 antisense 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual B Specificity Tests Dissociation analysis Sample disso
21. ollowing for 1 entire qPCR plate For 1 entire plate 1 750 ul 2X SYBR Green qPCR Mastermix buffer 20 ul LncRNA cDNA from Step A 1 730 ul RNase free water 3 500 ul Total Aliquot 29ul of Mastermix per well in your qPCR Plate Resuspend Primers in Primer plate with 44ul RNase free water per well before use the primers are dried down in the stock primer plate Then Load 2ul per well of each of the Primers from the Primer plate into your qPCR plate well A1 into qPCR plate A1 etc The Mastermix contents can be scaled up or down depending upon on your experimental needs If you want to perform the reactions in triplicate scale up the cDNA synthesis reactions by 3 fold and add 3X the RNA input Or simply follow the above recipe three times for each of the qPCR plates you want to run as replicates 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 96 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
22. rview For the last few decades of the 20th century the underlying dogma of molecular biology has been that the purpose of RNA is to direct the assembly of proteins from amino acids A few exceptions to this paradigm were known for example ribosomal RNA and transfer RNA which are functional RNA macromolecules that do not code for protein or viral genomes that exist as or pass through an RNA phase as part of total genome replication Non coding RNAs ncRNAs include the familiar housekeeping RNAs ribosomal transfer small nuclear and small nucleolar RNAs and the thousands of regulatory RNAs that are the subject of recent intense exploration Regulatory ncRNAs are arbitrarily classified by size small ncRNAs sncRNA being less than 200 bp and long ncRNAs IncRNA greater than 200 bp The sncRNAs include other sub classifications microRNA miRNA endogenous small inhibitor RNA endo siRNA and PIWI associated RNA piRNA The roles of IncRNAs in the regulation of gene expression and organismal development are diverse and just beginning to be discovered Biological processes dependent upon IncRNAs include imprinting and gene dosage regulation stem cell pluripotency embryonic development and segmentation hematopoiesis and neural cell fate determination LncRNAs may employ a number of mechanisms to impact gene expression via cis and trans processes Gene imprinting While the function of parental gene imprinting is still unclear IncR
23. so regulated by IncRNAs Some HOX related IncRNAs operate in cis having either enhancing or repressive effects However some like the human HOTAIR work in trans and may function as scaffolds for histone modifying complexes It is not clear if trans acting IncRNAs like HOTAIR are involved in the process of identifying the DNA sites to which the complexes will be recruited or if that function is retained by the protein elements of the complex Stem cell pluripotency The promoters of more than 100 IncRNAs are bound by stem cell factors Disruption of these IncRNAs can alter cell differentiation lincRNA RoR is involved in the reprogramming of fibroblasts back to a pluripotent state Thus IncRNAs are likely to play important roles in both normal development and processes that require maintenance of adult stem cell pools Cell fate determination LncRNAs are implicated in cell fate determination events in multiple cell lineages including the nervous system TUG1 is an IncRNA that may enhance rod gene expression and suppress cone gene expression in the developing eye Evf2 is a mouse IncRNA that appears to have both cis and trans effects to repress DIx5 DIx6 and Gad1 during forebrain development Dysregulated expression of IncRNAs has been shown to be associated with a broad range of diseases such as Alzheimer s psoriasis and many cancers Studying the expression patterns of IncRNAs will be a crucial method to understanding the roles they play in many
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