RNA-Quant cDNA synthesis kit User Manual
Contents
1. RNA 7 Spr Qu nt System Biosciences RNA Quant cDNA Synthesis Kit Profile any RNA by real time qPCR Cat RA430A 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 RNA Quant cDNA Synthesis Kit Cat RA430A 1 Contents l Introduction and Background A Overview B RNA Quant cDNA synthesis overview C List of components ll Protocol A RNA Quant cDNA reaction set up B Mastermix qPCR Reaction setup lll Sample Data and Quality Control A Profile mRNAs snRNAs snoRNAs and rRNAs B Profile microRNAs and IncRNAs IV Troubleshooting V RNATechnical References VI Technical Support VII Licensing and Warranty Statement 888 266 5066 Toll Free 650 968 2200 outside US ARON ooN 10 11 12 13 Page 1 System Biosciences SBI User Manual Introduction and Background A Overview Types of RNAs 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 These are the functions of messenger RNAs mRNAs mRNAs code for protein genes have 5 mG caps and most often have poly A tails A few exceptions to this paradigm were known for example ribosomal RNA and transfer RNA which are functi2. 7 stem loop precursors harbor features of RNase III cleavage products Nucleic Acids Res 31 6593 6597 Chomczynski P and Mackey K One hour downward capillary blotting of RNA at neutral pH 1994 Anal Biochem 221 303 305 Shi R Chiang V L 2005 Facile means for quantifying microRNA expression by real time PCR BioTechniques 39 519 525 Ding Y Chan C Y and Lawrence C E 2005 RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble RNA 11 1157 1166 Griffiths Jones S Grocock R J Van Dongen S Bateman A Enright A J 2006 miRBase microRNA sequences targets and gene nomenclature Nucleic Acids Research 34 D140 D144 Shingara J Keiger K Shelton J Laosinchai Wolf W Powers P Conrad R Brown D Labourier E 2005 An optimized isolation and labeling platform for accurate microRNA expression profiling RNA 71 1461 1470 He L Thomson J M Hemann M T Hernando Monge E Mu D Goodson S Powers S Cordon Cardo C Lowe S W Hannon G J Hammond S M 2005 A microRNA polycistron as a potential human oncogene Nature 435 828 833 Lai E C Wiel C Rubin G M 2004 Complementary miRNA pairs suggest a regulatory role for mIRNA miRNA duplexes RNA 10 171 175 Ambros V Bartel B Bartel D P Burge C B Carrington J C Chen X Dreyfuss G Eddy S R Griffiths Jones S Marshall M Matzke M Ruvkun G Tuschl T 2003 A uniform system for
3. License Use of the RNA Quant Kit 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 replace 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
4. 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 2012 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 13
5. synthesis kit that is designed to tag and convert any type of RNA into detectable and quantifiable cDNAs The system allows for the ability to quantitate dynamic fold differences of RNAs across 20 separate experimental RNA samples The RNA Nucleolus N ctcleu s Quant kit also includes 3 endogenous RNA reference qPCR assays as normalization signals These assays include miR 16 microRNA Y RNA IncRNA and GAPDH mRNA These three qPCR assays will work with both human and mouse RNA samples To ensure optimal results please read the entire manual before using the reagents and material supplied with this kit Profile any RNA no matter its identity or localization Page 2 ver 1 042412 www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 B RNA Quant cDNA synthesis overview How the RNA Quant Kit Works 10 pg 10 pg A Any RNA total RNA anchor tailed mRNAs microRNAs sn snoRNAs IncRNAs 5 rRNAs P Tag All RNAs eae polyA tailed 5 AAAAAAAAA 3 J IncRNAs Incubate at 37 C 30 min 2 Anneal Adaptor 5 AAAAAAAAA 3 NVTTTTTTTTT M 5 Anneal adaptor 60 C 5 min Sl AAAAAAAAA 3 Convert to cDNA 3 lt a TT TTT TTT es 5 RT random primers to make cDNA 42 C 60 min cDNA pool of anchor tailed RNAs 3 TTT TT TT a 5 cDNA ready for qPCR analysis The initial polyadenylation step greatly enhances cDNA synthesis yields of small RNAs over 100 fold and enables the
6. ancer 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 106 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual VI 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 info systembio com Technical Support tech systembio com Ordering Information orders systembio com Page 12 ver 1 042412 www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 vil Licensing and Warranty Statement Limited Use
7. l Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Then Load 1ul per well of each of the Control qPCR Primer 10 uM into your qPCR plate well miR 16 Y RNA or GAPDH C Mastermix qPCR Reaction Setup for 384 well qPCR plate To determine the expression profile for the RNAs under study mix the following for 1 well in a 384 well qPCR plate For 1 well in 384 well plate 6 ul reaction 3 ul 2X SYBR Green qPCR Mastermix buffer 0 06 yp RNA Quant cDNA from Step A 0 1 pl 3 Universal Reverse Primer 10 uM 2 6 ul RNase free water 5 7 ul Total Starting RNA amount and amount of cDNA per qPCR reaction Starting RNA input Amount of RQ cDNA to add per rxn 10pg 100 ng 0 1pI to 0 5pl 100ng 1 ug Dilute 1 500 gt 1 ug Dilute 1 5 000 Aliquot 5ul of Mastermix per well in your qPCR Plate Then Load 0 2ul per well of each of the Control qPCR Primer 10 uM into your qPCR plate well miR 16 Y RNA or GAPDH Page 6 ver 1 042412 www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 lll Sample Data A Profile mRNAs snRNAs snoRNAs and rRNAs Total RNA was harvested from human HT1080 cells using the following protocol Sample amplification plots and specificity tests using dissociation analyses are shown below Protocol per one well of 6 well plate Confluent cells in a 6 well remove media 1 2 Add 1ml Trizol directly to cells on plate 3 Incubate at Room temperature fo
8. measurement of microRNAs and the usage of small RNAs like U6 and RNU43 to be included as reference controls for qPCR analysis C List of components 5X Reverse Transcriptase Buffer Random i 20 ul Reverse Transcriptase 30 pl 0 1 M Dithiothreitol DTT 50 pl dNTP Mix RNase free Water 600 ul 3 Universal Reverse primer 50 ul miR 16 microRNA qPCR assay 10M human and mouse 50 ul Y RNA IncRNA qPCR assay 104M human and H mouse MOUS mRNA qPCR assay 104M human and mouse 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual 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 SBI recommends using the following SYBR Green reagents e 2X Maxima SYBR Green with Rox Cat K0223 from Fermentas highly recommend e Power SYBR Master Mix Cat s 4368577 4367650 4367659 4368706 4368702 4368708 4367660 from Applied Biosystems Page 4 ver 1 042412 www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 ll Protocol A RNA Quant cDNA reaction setup for 1 RNA sample to be assayed on qPCR 96 well plates It is important to start with total RNA that includes the IncRNA fraction RNA input can be as low as 1 2 ug total For optimum signals perform the following EB Dilute your RNA to 200 400 ng ul Start i In a thin walled PCR tube or s PCR compatible
9. microRNA annotation RNA 9 277 279 Obernosterer G Leuschner P J F Alenius M Martinez J 2006 Post transcriptional regulation of microRNA expression RNA 12 1 7 Dostie J Mourelatos Z Yang M Sharma A Dreyfuss G 2003 Numerous microRNPs in neuronal cells containing novel microRNAs RNA 9 180 186 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 Disease 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 C
10. onal 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 There are now numerous exceptions that include microRNAs small nuclear RNAs snRNAs small nucleolar RNAs snoRNAs and long non coding RNAs IncRNAs The functions of each class of RNA are briefly summarized in the Table below Type of RNA Function Messenger RNAs mRNAs Code for Proteins MicroRNAs miRNAs Regulate mRNA translation Bridge splicing events during Small Nuclear RNAs ate transcription via the snRNAs spliceosome Small Nucleolar RNAs Guide methylation on target snoRNAs RNAs Bind chromatin for epigenome changes and scaffolds for protein complexes Structural and catalytic subunits of ribosomes Long Non coding RNAs IncRNAs Ribosomal RNAs rRNAs 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 This manual provides details and information necessary to use the RNA Quant cDNA
11. plate well combine 5 ul Total RNA 1 2 p9 STEP 1 2 ul 5X PolyA Buffer 1p 25mM MnCl PolyA Tail 1 5 pl 5mM ATP 0 5 ul PolyA Polymerase 10 ul Incubate for 30 min at 37 C Add 0 5 pl Oligo dT Adapter STEP 2 Heat for ne at 60 C Anneal Anchor Let cool to room temp for 2 min dT Adapter Add 4 ul 5X RT Buffer 2 ul dNTP mix STEP 3 1 5 pl 0 1M DTT Synthesize 1 5 pl Random Primer mix cDNAs 1 ul Reverse Transcriptase 20 5 ul Total in tube Incubate for 60 min at 42 C Heat for 10 min at 95 C The RNA Quant cDNAs can be stored at 20 C For more sensitive applications a single D Oo n e 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 B Mastermix qPCR Reaction Setup for 96 well qPCR plate To determine the expression profile for the RNAs under study mix the following for 1 well in a 96 well qPCR plate For 1 well in 96 well plate 30 ul reaction 15 ul 2X SYBR Green qPCR Mastermix buffer 03 ul RNA Quant cDNA from Step A 0 5 wl 3 Universal Reverse Primer 10 uM 14 7 ul RNase free water 29 ul Total Starting RNA amount and amount of cDNA per qPCR reaction Starting RNA input Amount of RQ cDNA to add per rxn 10pg 100 ng 0 5ul to ipl 100ng 1 ug Dilute 1 100 gt 1 ug Dilute 1 1 000 Aliquot 29u of Mastermix per well in your qPCR Plate 888 266 5066 Tol
12. r 5 minutes for complete lysis 4 Collect Trizol cell mixture and transfer to 1 5ml tube 5 Add 200 ul Chloroform vortex 15 seconds 6 Centrifuge mixture for 15 minutes at 4 C 7 Collect aqueous layer and transfer to fresh 1 5 ml tube 8 Add equal volume 250 ul Isopropanol mix by inversion 9 Precipitate RNA overnight at 20 C 10 Centrifuge at 13 000 rpm for 20 minutes 11 Remove supernatant 12 Wash 1X with 500 ul 80 Ethanol 13 Centrifuge again for 5 minutes at 13 000 rpm 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 RNA Quant cDNA synthesis 2ug svete Eat 18S rRNA min A C 18SrRNA EE U6 snRNA f k Le Tm 73 C me 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual B Profile microRNAs and IncRNAs The same cDNA from IIIA above was used to profile microRNAs and IncRNAs by qPCR Sample amplification plots and specificity tests using dissociation analyses are shown below Angie Pict miR 18a MicroRNAs Rotisiins Spe colon et Z mensi sawon LncRNAs HOTAIR Page 8 ver 1 042412 www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 C Clean assay design with no background The poly A tailing and tagging method in the RNA Quant system generates cDNA Zero background that is highl
13. se of the amplification curve Also see the User Manual for your specific instrument or phone their technical support team for guidance Page 10 ver 1 042412 User Manual www systembio com RNA Quant cDNA Synthesis Kit Cat RA430A 1 V RNA Technical References selected Sonthelmer E J Carthew R W 2005 Silence from within Endogenous siRNAs and miRNAs Cell 122 9 12 Zamore P D Haley B 2005 Ribo gnome The big world of small RNAs Science 309 1519 1524 Bartel D 2004 MicroRNAs Genomics Biogenesis Mechanism and Function Cell 116 281 297 Kim Narry V 2005 Small RNAs Classification Biogenesis and Function Mol Cells 19 1 15 Valencia Sanchez MA Liu J Hannon GJ Parker R 2006 Control of translation and mRNA degradation by miRNAs and siRNAs Genes Dev 20 515 525 Lewis B P Burge C B Bartel D P 2005 Conserved seed pairing often flanked by adenosines indicates that thousands of human genes are microRNA targets Cell 120 15 20 Xie X Lu J Kulbokas E J Goulub T R Mooth V Lindblad Toh K Lander E S and Kellis M Systematic discovery of regulatory motifs in human promoters and 3 UTRs by comparison of several mammals Nature 434 338 45 Lagos Quintana M Rauhut R Lendeckel W Tuschl T 2001 Identification of Novel Coding for Small Expresses RNAs Science 294 853 858 Basyuk E Suavet F Doglio A Bordonne R Bertrand E 2003 Human let
14. y unique and when used in minus RT Controls with the 3 Universal reverse primer does not produce any signals in minus RT controls and does not detect any residual genomic DNA 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 IncRNA_ and microRNA qPCR 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 qPCR assays do not show any amplification signals even with spiked in genomic DNA Profile with confidence and only detect your RNAs of interest Assays do not _ detect Genomic DNA 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI IV Troubleshooting Problem Possible Solution Too much background in qPCR signals Use much less cDNA in the SYBR Green Mastermix No qPCR signals Did you select SYBR Green as the Detectors 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 for Ct analysis Typically place the threshold setting in the center of the exponential pha
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