Disease-related human lncRNA profiler User Manual
Contents
1. e Reverse transcription Kit e SYBR Green qPCR Master Mix e Real time qPCR Instrument e Instrument specific optical qPCR plates if you choose RA920D 1 e Calibrated Multi Channel Pipettor e RNase DNase free pipette tips and tubes 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual ll Protocol Please read through this entire protocol before beginning your experiment Prepare a workspace free of DNA contamination Wear gloves and maintain an RNase free work area while performing this protocol A RNA preparation and quality control High quality RNA is essential for obtaining good real time PCR results RNA samples must meet the standards of integrity and purity from protein organics and genomic DNA contamination For best results from the PCR Array all RNA samples should demonstrate consistent quality You can measure your RNA concentration and purity by UV spectrophotometry You can also check ribosomal RNA band integrity by electrophoresis or by using an Agilent BioAnalyzer e A260 A230 ratio should be greater than 1 7 e A260 A280 ratio should be between 1 8 and 2 0 e The total RNA concentration by A260 should be greater than 40 ug ml Eliminating genomic DNA contamination is essential for obtaining optimal real time gene expression profiling The problem associated with genomic DNA contamination is particularly acute for genes with low expression levels Even a few cop2. 7291 1071 6 Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a Yap KL et al Mol Cell 2010 Jun 11 38 5 662 674 Long non coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15 INK4B tumor suppressor gene Kotake Y et al Oncogene 2011 Apr 21 30 16 1956 62 Epub 2010 Dec 13 Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA Yu W et al Nature 2008 Jan 10 451 7175 202 206 MALAT 1 a novel noncoding RNA and thymosin beta4 predict metastasis and survival in early stage non small cell lung cancer Ji P et al Oncogene 2003 Sep 11 22 39 8031 41 Regulation and expression of a growth arrest specific gene gas5 during growth differentiation and development Coccia EM et al Mol Cell Biol 1992 Aug 12 8 3514 21 GAS5 a non protein coding RNA controls apoptosis and is downregulated in breast cancer Mourtada Maarabouni M et al Oncogene 2009 Jan 15 28 2 195 208 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual 11 14 15 16 17 18 19 Noncoding RNA gas5 is a growth arrest and starvation associated repressor of the glucocorticoid receptor Kino T et al Sci Signal 2010 Feb2 3 107 ra8 Large intergenic non coding RNA RoR modulates reprogramming of human induced pluripotent stem cells Loewer S et al
3. Nat Genet 2010 Dec 42 12 1113 7 Epub 2010 Nov 7 Erratum in Nat Genet 2010 Dec 42 12 3 p following 1117 Expression of a noncoding RNA is elevated in Alzheimer s disease and drives rapid feed forward regulation of beta secretase Faghihi MA et al Nat Med 2008 Jul 14 7 723 30 Epub 2008 Jun 29 lincRNAs act in the circuitry controlling pluripotency and differentiation Guttman et al Nature 2011 Aug 28 477 7364 295 300 doi 10 1038 nature10398 Chromatin signature reveals over a thousand highly conserved large non coding RNAs in mammals Guttman M et al Nature 2009 Mar 12 458 7235 223 7 Evolution and functions of long noncoding RNAs Ponting CP Oliver PL Reik W Cell 2009 Feb 20 136 4 629 41 Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real time PCR Galiveti CR et al RNA 2010 Feb 16 2 450 61 Epub 2009 Dec 29 Diagnostic and prognostic signatures from the small non coding RNA transcriptome in prostate cancer Martens Uzunova ES et al Oncogene 2011 Jul 18 doi 10 1038 onc 2011 304 Epub ahead of print Extensive and coordinated transcription of noncoding RNAs within cell cycle promoters Hung T et al Nat Genet 2011 Jun 5 43 7 621 9 doi 10 1038 ng 848 Page 22 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 VI Technical Support For more information about SBI products and to download manuals
4. 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 Road Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail tech systembio com General Information Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free _ 650 968 2200 outside US Page 23 Vil System Biosciences SBI User Manual Licensing and Warranty Statement Limited Use License Use of the Disease related human IncRNA Profiler 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
5. outlined herein SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Limited Warranty 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 Page 24 ver 1 100611 www systembio com
6. 2 Note In the presence of genomic DNA a PCR product of GDC in size of 184 bp is obtained M A B 400bp 300bp 200bp 100bp A RNA sample with genomic DNA contamination sample free of genomic DNA Page 10 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 For Customers who choose RA920D 1 a Resuspend primers in primer plate with 20 ul nuclease free water per well before use The primers are desicated in the primer plate Cocktail preparation in a reservoir OX SYBR Green qPCR Mastermix buffer cDNA 100 ul Nuclease free water 890 ul Total 2 090 ml C Using an eight channel pipette add 19 ul of cocktail to each well except well H12 the GDC in qPCR plate which matches your qPCR instrumentation Add 1ul NRT no RT sample or RNA sample 10u 2X SYBR Green qPCR Mastermix buffer 8 Nuclease free water into well H12 to detect genomic DNA contamination Load 1 ul per well of each of the primers from the primer plate into your qPCR plate Once reagents are loaded into the wells cover the plate with an optical adhesive seal 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 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 2 Real time qPCR Inst
7. 23948 ANRIL anti NOS2A 1 BCO17743 43430 200 BCMS BIC CCND1 B ANCR CMPD DD3 DGCR5 DISC2 DLG2AS EGO GASS GOMAFU H19 H19 AS HARIA HARIB HOTAIR HoTAIRM1 HOXALAS HOXA3AS HOXAGAS ioyaiias IPW IGF2AS AA489505 823151 BE873349 AK092154 LincRNA LincRNA LOC285194 LUST MALATI D KRASP1 L1PA16 MEG3 11 NEAT NCRMS PSF NDM29 PANDA PARS PCAT 1 PCAT 14 PCAT 29 PCAT 32 PCAT 43 PCGEM1 PR AT2 PRINS inhibiting RNA F PTENP1 RMRP ROR SAF SCA8 Sox20T SRA ST7OT1 ST70T2 ST7OT3 ST70T4 er G tmevpai 0 001762 1 WTI AS y Ys ZEB2NAT zsk Negative 9 control H 751 scRNA 5 8S rRNA 087 scaRNA U6 smRNA ACTB B2M PGK1 GAPDH HPRT1 RPL1A RPL13A GDC M M M i i small RNA transcripts housekeeping genes Internal Controls Page 6 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat 4RA920A D 1 Shipping Conditions The Disease related human IncRNA Profilers are shipped at Room Temperature or Blue Ice Storage Conditions Keep plates at 20 C for long term storage When stored at 20 their quality is guaranteed for 12 months The primers for the specific IncRNAs are provided desiccated in the wells of the optical qPCR plates D Additional Required Materials e RNA Isolation e Nuclease free
8. SSBI System Biosciences Disease Related Human LncRNA Profiler Cat RA920A B C D 1 User Manual Store at 209C 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 ver 1 100611 contained in this user manual Disease related human IncRNA Profiler Cat RA920A D 1 Table of Contents l Introduction and 2 A OVEMNVIOW 2 Long noncoding RNAs and 2 C Overview of the Entire Protocol 5 D Additional Required 5 7 12 udeeci rer 8 A RNA preparation and quality control 8 B First Strand cDNA Synthesis esee 9 C Real time qPCR Reaction 9 D Data Analysis AACt 12 Hl Quality Control and Sample Data 14 A Validatiori Tests ctore ee eerte 14 B ee e ee ee RE 19 IV Troubleshooting orte rient eerta 20 V References tei a 21 VI Technical Support eese ener 23 VII Licensing and Warrant
9. e level of genomic DNA contamination is too low to affect gene expression profiling results No action is needed If the value is less than 35 then genomic DNA contamination is evident 3 Calculate the ACt for each IncRNA in the plate ACt Internal Control 4 Calculate the AACt for each IncRNA across two PCR Arrays AACt ACt Sample ACt control 5 Calculate the fold change for each gene from the control to the sample as 272 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Ill Quality Control and Sample Data A Disease related human IncRNA Profiler Validation Tests 1 Real time qPCR Validation The Disease related human IncRNA Profiler plate was tested using a cohort of 9 cell line samples 293T HT1080 hES HDF HFF MCF7 MCF10A MDA MB 231 mesenchymal stem cells converted to cDNA using the ProtoScript amp M MuLV Taq RT PCR Kit The resulting cDNA was tested according to aforementioned protocol Shown below are the results of Real time amplification plot for the entire plate for selected cell lines Page 14 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 2 End point PCR Validation The Disease Related human IncRNA Profiler plate was tested using a cohort of 9 cell line samples converted to cDNA using the ProtoScript amp M MuLV Taq RT PCR Kit 1ug of RNA was used to synthesize first strand cDNA w
10. e of correlation R gt 0 98 40 35 Ri 30 X 25 E 20 3 15 10 5 0 0 5 10 15 20 25 30 35 40 Scientist B 40 35 30 25 2 20 3 15 10 5 0 0 5 10 15 20 25 30 35 40 Scientist 888 266 5066 Toll Free _ 650 968 2200 outside US Page 17 System Biosciences SBI User Manual 5 Specificity Tests A single IncRNA specific product for each primer set was amplified by disease related human IncRNA profiler 21A AAA1 aHIF AK023948 ANRIL Anti NOS2A BACE1 BC017743 BC043430 BC200 BCMS BIC AK023 Anti 1 BCO17 BC043 21A 1 aHIF ANRIL as PC200 BCMS BIC 400bp 300bp 200bp 100bp Page 18 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 B Sample Data analysis of disease related human IncRNA expression levels in different cell lines using Real time qPCR The cDNAs were synthesized from both 293T and HT1080 cells The signals were normalized to expression levels of housekeeping genes and small RNA transcripts ACt increase and decrease in 293T vs HT1080 cells are graphed 20 HT1080 293T 10 5 4 0 4 DT T o Lm o a u ACt Value lt a da ttqqudaa A d OG O G D G G r r D o 4 44 lt lt d Li 888 266 5066 Toll Fr
11. ed mixture of hexamers and Oligo d T 23VN primer which provides even and consistent coverage of the RNA template population across a wide range of RNA template concentration Based on your experiments design you can choose either Oligo dT primer random primer mix or strand specific primer to synthesis your cDNA C Real time qPCR Reaction Setup 1 Mastermix qPCR Reaction Setup for a 96 well qPCR plate a Briefly spin down all reagents b Mix the following components in a 5 ml tube or reservoir 2X SYBR Green qPCR Mastermix buffer 1 1 ml cDNA 100 ul Nuclease free water 1 0 ml Total 2 2 ml 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Load the Disease related human IncRNA Profiler e Carefully remove the profiler array from its sealed bag e Add 20ul of cocktail to each well except well H12 the GDC in array plate from a reservoir with an eight channel pipette e Add 1ul NRT no RT sample or 1 RNA sample 10 2X SYBR Green qPCR Mastermix buffer 9j Nuclease free water into well H12 to detect genomic DNA contamination Note This recipe provides an excess volume of 200 ul for the 96 well format to allow for multiple pipetting Note Change pipet tips following each addition to avoid any cross contamination between the wells or reactions Note Maxima SYBR Green Rox qPCR Master Mix 2x was used for in house testing Thermo Scientific Cat K022
12. ee 650 968 2200 outside US Page 19 System Biosciences SBI User Manual IV Troubleshooting Problem Possible Solution Too much background in Use much less cDNA in the SYBR qPCR signals Green Mastermix No qPCR signals Did you select SYBR Green as the Detectors Reporter Dye Did the internal control work Use more cDNA in Mastermix Check Mastermix contents and try a subset with internal control as a positive control Also try lowering the Annealing Temperature to 50 C How do select the Threshold Typically place the threshold setting level for Ct analysis in the upper third of the exponential phase of the amplification curve Also see the User Manual for your specific instrument or contact their technical support team for guidance Page 20 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 V 1 10 References IncRNAdb a reference database for long noncoding RNAs Amaral PP et al Nucleic Acids Res 2011 Jan 39 Database issue D146 51 Epub 2010 Nov 25 Long noncoding RNAs and human disease Wapinski O Chang HY Trends Cell Biol 2011 Jun 21 6 354 61 Epub 2011 May 6 A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression Wang et al Nature 2011 Apr 7 472 7341 120 4 Epub 2011 Mar 20 Long non coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis Gupta RA et al Nature 2010 Apr 15 464
13. egulates BACE1 levels in response to a variety of stresses acting as a post transcriptional regulator Consistent with BACE1 expression BACE1AS expression were found increased in subjects with AD as well as in amyloid precursor protein transgenic mice In summary it has been shown that dysregulation of IncRNAs contributes to numerous diseases highligting their potential use as biomarkers and therapeutic targets in the future However there Page 4 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 are still many missing dots in our current understanding of IncRNA function Future studies are needed to elucidate the mechanism by which IncRNAs are dysregulated thereby contributing to the pathogenesis of disease Disease related human IncRNA Profiler is your first step toward the understanding of the role of IncRNAs in human diseases C Overview of the Entire Protocol Isolate RNA from your experimental samples 1ug RNA is recommended treat with DNase 4 Convert Total RNA to cDNA E cDNA1 cDNA2 Add cDNA to qPCR master mix vs vs Aliquot mixture across PCR array Array profiles the expression of 83 disease related IncRNA plus controls T Perform real time PCR Collect real time PCR data using your instrument s software Profiler 1 Profiler 2 aeos Analyze Changes in IncRNA expression Straightforward analysis Simply cut and paste the Ct value collec
14. ies of genomic DNA contamination can significantly alter the interpretation of RT PCR results for low copy mRNA Due to the presence of pseudogenes even cross intron primers are not a reliable method for avoiding amplification resulting from contaminating genomic DNA The Genomic DNA Control GDC in this PCR Array specifically tests for genomic DNA contamination in each sample during each run Researchers can easily detect potential genomic DNA contamination using the representative Ct value for the GDC well on the PCR Array A GDC threshold cycle value less than 35 indicates the presence of a detectable amount of genomic DNA contamination that is recommended to be addressed Page 8 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 Greater amounts of input total RNA yield a greater number of positive results Lower amounts of input total RNA yield a smaller number of positive results and increase false negative results B First Strand cDNA Synthesis Use the same amount of total RNA in this reaction for every sample We recommend that first time users start with 1 0 2 0 ug of total RNA for 96 well plate format Please follow the protocol of the cDNA synthesis provided by the manufacturer Dilute the resulting cDNA in 1004 with nuclease free H2O For in house testing ProtoScript M MuLV Taq RT PCR Kit NEB Cat E6400S was used for cDNA synthesis with Random Primer Mix Random Primer Mix is a optimiz
15. ith Random Primer Mix The resulting cDNA was tested according to the protocol Shown below are the end products for the entire plate selected from hES cell 293T and HT1080 cells and separated on a 1 5 agarose gel and stained with ethidium bromide Mi 23 4 5 6 7 8 9 10 11 12 400bp A 100bp 400bp 100bp 300bp 100bp 400bp D 100bp 400bp E 100bp 400bp 100bp 400bp G 100bp 400bp H 100bp 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual 3 Sensitivity Tests The cDNAs were synthesized using decreasing amounts of total starting RNA input from human ES cells Ct values were determined using the software automatic baseline and Ct settings ROR NEAT1 35 35 30 30 25 o 25 5 20 2 20 g s 2 15 n 9 10 5 5 0 o 1000 500ng 100ng 50ng 1000ng S00ng 100ng 50 Input RNA Input RNA 21A GOMAFU 35 35 30 30 25 25 5 20 S 2 S as E 15 5 10 5 5 0 o 1000 500ng 100ng 50 1000ng 500ng 100ng 50 Input RNA Input RNA Page 16 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 4 Reproducibility Tests Two sets of raw Ct data were obtained by two different scientists A amp B at two different times using two different cell lines on Disease related human IncRNA Profiler and are directly compared The results demonstrate a high degre
16. nd establishing repressive epigenetic mark Studies from Yu W et al and Kotake Y et al have also shown that overexpression of ANRIL in prostate cancer results in the silencing of INKAn ARF INK4a and p15 CDKN2B by heterochromatin formation Different from oncogenic IncRNAs such as HOTAIR ANRIL and MALAT1 IncRNA GAS5 growth arrest specific 5 represents an example as a tumor suppressor Studies from Coccia EM and Mourtada Maarabouni M group have shown that GAS5 can be induced under starvation conditions and is abundant is abundant in cells whose growth has been arrested In addition GAS5 has also been observed to be downregulated in breast cancer Kino T et al revealed that Gas5 bound to the DNA binding domain of the glucocorticoid receptor GR by acting as a decoy glucocorticoid response element GRE thus competing with DNA GREs for binding to the GR Function as a riborepressor of the GR GAS5 influences cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR In addition to cancer IncRNAs have also been shown involved in other disease such as Alzheimer s Disease AD A study from Faghihi MA et al nicely demonstrated how IncRNA BACE1 AS involves in the pathogenesis of AD B secretase 1 BACE1 is a crucial enzyme in AD pathophysiology which has elevated level in subjects with AD compared with normal controls As a natural antisense transcript BACE1AS rapidly and reversibly upr
17. rument Parameters Follow the guidelines as detailed for your specific Real time instrumentation The following parameters tested by SBI were performed on an Applied Biosystems 7900 Real time PCR System but can also apply to an ABI 7500 or 7300 system Instrument Setup qPCR cycling program 1 50 C 2 min 2 95 C 10 min 3 95 C 15sec 4 60 1 min 40 cycles of steps 3 4 An additional recommendation is to include a melt analysis 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 Thresholds should be set within the exponential phase of the amplification plots with software automatic baseline settings D Data Analysis AACt Method Access our free Disease related human IncRNA Profiler data analysis web portal from the following address http www systembio com Incrna research long non coding rna literature 1 Change all Ct values reported as greater than 35 or as N A not detected to 35 At this point any Ct value equal to 35 is considered a negative result Page 12 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 2 Examine the Threshold Cycle values of the Genomic DNA Control wells If the value is greater than 35 then th
18. t al This repressive action is mediated by the interaction of HOTAIR with the Polycomb Repressive Complex 2 PRC2 Later study from John s group further found that HOTAIR is dramatically overexpressed in breast tumors and the expression of HOTAIR in primary breast tumors is a powerful prognosis marker of patient outcomes such as metastasis and death Enforced expression of HOTAIR causes altered H3K27 methylation pattern and increased invasiveness In contrast the depletion of HOTAIR results in the opposite cellular phenotype These studies demonstrate how IncRNA manipulate the epigenetic machinery to remold the epigenetic landscape leading to cancer Recent genome wide studies have revealed that numerous protein coding mRNAs have natural antisense transcript partners most of which seem be noncoding RNA Despite the lincRNAs such as HOTAIR dysregulation of antisense ncRNAs could also lead to cellular transformation Antisense ncRNA ANRIL is one of the examples ANRIL is transcribed as antisense to the INKAn ARF INK4a promoter and overlaps with two exons of p15 CDKN2B It controls expression in the INK4A ARF locus 888 266 5066 Toll Free _ 650 968 2200 outside US Page 3 System Biosciences SBI User Manual which comprises the tumor suppressor genes INK4n ARF INK4a p16 CDKN2A and p15 CDKN2B As demonstrated by Yap et al ANRIL interacts with CBX7 resulting in the targeting of the polycomb repressive complex 1 PRC1 to the chromatin a
19. ted by your real time instrument into an analysis spreadsheet Fold change IncRNA expression between your samples are automatically calculated Sample A 9 15 Sar 5 Sample B S 10 24 r r o oeugdgasaggugesoseosSso0oudusmunsceusdsd 4 dcG d amp d amp amp amp O O O O O O O dH H OQ tu i i LU cn d Xx 2 5 Uu 888 266 5066 Toll Free _ 650 968 2200 outside US Page 5 System Biosciences SBI User Manual List of Components The Disease related human IncRNA Profiler is available different plate formats each tailored to a specific subset of real time PCR instruments RA920A 1 ABI 7000 7300 7500 7900HT 96 well RA920B 1 ABI 7500 Fast 7900HT Fast StepOnePlus 96 well 2 2 Bio Rad Opticon Opticon2 Chromo 4 RA920C 1 iCycler MyiQ iQ5 CFX96 96 well 2 2 Eppendorf MasterCycler ep RelPlex RA920D 1 Any Real Time Instruments 96 well 20 a All 83 IncRNAs chosen for the array are based on publications and they are implicated in diseases ranging from neurodegeneration to cancer The array plate also includes the housekeeping genes and small RNA transcripts which belongs to small nuclear RNA small cytoplasmic RNA and small cajal body specific RNA for normalization purpose The Genomic DNA Control GDC in each PCR Array specifically tests for genomic DNA contamination in each sample during each run 1 2 3 4 5 6 7 8 9 10 11 12 21 1 aHIF AK0
20. using the materials supplied with this kit B Long noncoding RNAs and diseases A distinctive feature of eukaryotic genomes is the abundance of noncoding RNA ncRNAs In addition to small regulatory RNAs such as microRNAs genomic projects over the past decade have revealed the existence of long noncodingRNAs IncRNAs those more than 200 bp in length including large intergenic ncRNA lincRNA and antisense ncRNAs Like microRNAs the expression Page 2 ver 1 100611 www systembio com Disease related human IncRNA Profiler Cat RA920A D 1 of many identified IncRNAs shows spatial and temporal specific patterns Recent studies also demonstrate that LncRNAs participate in a diverse range of biological processes from cell cycle control to cell differentiation through distinct mechanisms at the transcriptional post transcriptional and epigenetic level Almost every step in the life cycle of genes can be influenced by IncRNAs The involvement of IncRNAs in human diseases provides a great opportunity for biomedical research and their clinical implications could be far more prevalent than we previously imagined Like protein coding genes IncRNAs can serve as oncogenic and tumor suppressor genes The story of HOTAIR illustrates nicely how the relationship between epigenetic regulation by IncRNA and cancer HOTAIR was first identified as a 2 2kb IncRNA in the HOXC locus which represses transcription in trans of HOXD genes by John Rinn e
21. y Statement 24 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction and Background A Overview Long noncoding RNAs IncRNAs as a new class of transcripts have been recently revealed to be pervasively transcribed in the genomes of human and mouse Multiple lines of evidence increasingly link IncRNAs to diverse human diseases Disease related human IncRNA profiler qPCR array allows for the quantification of differential expression of 83 individual IncRNAs among various experimental RNA samples All 83 IncRNAs chosen for the array are based on publications and they are implicated in diseases ranging from neurodegeneration to cancer The array plate also includes the house keeping genes and small RNA transcripts for normalization purposes Using real time RT PCR you can easily and reliably analyze expression of a panel of IncRNAs that have potential roles in a variety of cellular processes such as cell cycle progression differentiation self renewal and apoptosis Disease related human IncRNA profiler is easy convenient sensitive and specific It is suitable for cDNA synthesized by either random primer oligo dT primer or strand specific primer It can be used for research in cancer stem cells immunology biomarker discovery amp validation as well as phenotypic analysis of cells To ensure optimal results please read the entire manual before
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