Global MicroRNA Amplification Kit
Contents
1. 181a 142 Fig 3 Maintenance of relative expression levels of RNA amplified using the Small RNA Amplification and Cloning kit In this experiment 50 ng of a pool of RNAs from 4 different tissues was amplified and tested for the presence of 10 different noncoding small RNA by RT PCR The same RNA pool was tested and quantitated for the same small noncoding RNA before amplification Care was taken to avoid the plateau phase of the PCR amplification For all 10 small RNAs the relative levels were maintained before and after amplification D Overview of Protocol Page 4 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A 1 Ligation 1 hr 3 on 5 RT and adaptor attachment 4 min won ww m y 5 melt destroy RNA 10 min 95 C 17 PCR 1 hr gt ems sO sec PCR extension time TZ 3i biotinylated primer amplified OR M cDNA T7 IVT with labeled nucleotides Sinn 3 5 VAFVAPVAPVNAVAA 3 5 Any 3 sense 5 AAAAARARARAN 3 RNA 5 AAAPAPVAPARA 3 M sense RNA il qRT PCR microRNA Microarrays Fig 4 Small RNA Amplification Procedure 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual E List of Components Each Small RNA Amplification and Cloning Kit provides enough material to amplify small RNA from 10 different RNA samples 10 reactions 8 yu Ligase Cocktail T4 DNA amp RNA ligase2. on ice The first strand cDNA can be stored at 20 C until you are ready to proceed with the Second Strand cDNA Synthesis and Amplification D Second Strand cDNA Synthesis and Amplification 1 To each first strand synthesis reaction from Part C add the following 77 ul RNase free Water 10 ul 10X PCR Buffer 2 ul dNTP Mix 4 yu 3 Adaptor Primer 4 ul 5 Adaptor Primer 3 ul PCR Polymerase 100 ul Total volume including the 10 ul from Part C 2 Placethe reactions in a thermal cycler and cycle using the following program 95 C for 5 min 95 C for 25 sec The of cycles depends on the 55 C for 20 sec amount of total RNA used in the 72 C for 30 sec ligation reaction 72 C for 30 sec 15 C hold Note You will need to vary the number of cycles depending on the amount of starting RNA Refer to the table below to determine the approximate number of times you should cycle Starting RNA Cycles 500 ng 33 100 ng 36 20 ng 39 5 ng 42 3 After amplification run 2 5 ul of each reaction on a 2 5 3 5 agarose gel in 1X TBE or TAE Buffer Include a DNA size ladder with markers in the range of 50 2 000 bp e g Bio Rad AmpliSize DNA Ladder You should see results similar to those shown in Figure 5 The typical yield from 50 ng RNA and above is 2 3 ug cDNA from 5 ng the yield is 1 1 5 ug 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Depending on your particu
3. 1 If desired the amplified cDNA can be cloned by standard protocols for DNA sequence analysis For miRNA microarray studies the amplified cDNA is first purified on spin column and labeled via T7 in vitro transcription in the presence of fluorescently labeled nucleotides The size of the amplified cDNA can be adjusted by the amount of time the reverse transcription is conducted Shorter reaction times will enrich for smaller RNA amplification Under conditions suggested in the user manual 5 minute RT reaction the size range of x 500nt RNA is efficiently amplified The size distribution and banding pattern of amplified cDNA is shown in Figure 1 below amplified cDNA bp 500 400 200 region of EG mature microRNA together with adaptor Fig 1 Size distribution of amplified cDNA using the Small RNA Amplification and Cloning Kit In this study 50 ng of a pool of RNAs from 4 different tissues was amplified with the SBI kit and a small portion examined by gel electrophoresis A banding pattern was observed which is consistent with amplification of several highly abundant small RNAs as indicated Because of the very small size and heterogeneity of microRNAs 19 24 nucleotides and low abundance it is not possible to observe the microRNA However as shown in Figure 2 the presence of microRNA is clearly visible The yield of amplified cDNA is sufficient for several hundred quantitative PCR experiments or several microRNA micro
4. 70 yu Ligation Buffer 15 ul Control RNA 25 ng ul 1 2 ml RNase free Water 10 ul 3 Adaptor 60 ul 3 Adaptor Primer 10 ul 5 Adaptor 50 l 5 Adaptor Primer 20 ul Reverse Transcriptase 80 ul 5XReverse Transcriptase Buffer 30 ul Dithiothreitol DTT 50 gu dNTP Mix 150 ul 10X PCR Buffer 35 l PCR Polymerase Proof reading The kits are shipped in blue ice and should be stored at 20 C upon receipt Properly stored kits are stable for 1 year from the date received F Additional Required Materials Thermocycler with heated lid 2 5 3 5 Agarose Gel in Tris Borate EDTA TBE or Tris Acetate EDTA TAE Buffer DNA Size Ladder with markers from 50 to 2 000 bp Bio Rad AmpliSize DNA Ladder Cat 170 8200 Optional for samples from sources with high RNase activity Ribonuclease Inhibitor Ambion SUPERase IN Cat 2694 Page 6 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A 1 G Procedural Guidelines e Before dispensing completely thaw all reagents Vortex to mix thoroughly all reagents except for the enzymes After adding reagents to the mixture pipette up and down 5 10 times to ensure mixing e Briefly centrifuge each mixture once all the components have been added to ensure there are no reagents left on the sides of the tube separated from the reaction mixture e When setting up multiple reactions we recommend you prepare a master mix e Both first and second strand cDNA is rel
5. C for 1 hour and then keep on ice until the RT step Step C The ligation reactions can be also be stored at 70 C and used later Page 8 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A 1 C First Strand cDNA Synthesis 1 For each RNA sample add the components to a 0 2 or 0 5 ml PCR tube in the order specified 4 0 ul RNase free Water 0 5 ul 3 Adaptor Primer 1 0 ul Ligation from Step B 5 5 ul Total volume 2 Incubate the reactions at 65 C for 1 minute and then place them at 42 C for 5 minutes Then keep reactions on ice 3 While the reactions are incubating set up a Master Mix sufficient for the number of first strand synthesis reactions you are processing This is done by adding to a microfuge tube the volume of each of the following components multiplied by the number of reactions you are processing 2 0 ul 5X Reverse Transcriptase Buffer 1 0 ul dNTP Mix 0 5 ul 5 Adaptor 0 5 ul Dithiothreitol DTT 0 5 ul Reverse Transcriptase 4 5 ul Total volume Note If you have 2 reactions you should have 9 ul of Master Mix if you have 3 reactions 13 5 ul etc 4 After incubating each of the first strand reactions from step 2 for 5 minutes at 42 C keep on ice until you are ready to start the RT step Add 4 5 ul of the Master Mix set up in step 3 5 Incubate the first strand reactions for 5 minutes at 42 C and then place the tubes at 95 C for 10 minutes Keep reactions
6. of 2 10ug of total RNA For the study of low abundant miRNAs by microarrays it can be necessary to size select for low molecular weight RNAs before labeling The need for microgram amounts of RNA places severe restrictions on the types of tissue that can be studied thus excluding clinical tissues such as FFPE or small biopsies or RNA obtained from laser capture microdissection in which yields of RNA can be in the low nanogram range C Small RNA Amplification The current method for amplifying miRNA involves many steps and can take several days to complete The method requires size selection of small RNA two ligation steps each requiring gel electrophoresis and RNA extraction steps an RT step followed by PCR amplification SBI s small RNA amplification system includes 3 steps which can be completed in less than 4 hours 1 Ligation of an adaptor to the 3 end of the RNA Our use of a chimeric DNA RNA adaptor and unique cocktail of enzymes and reaction conditions ensures efficient ligation 2 Reverse transcription of the RNA and attachment of a 5 end adaptor in the same reaction by template switching 3 PCR amplification of the cDNA with a PCR polymerase mixture that incorporates a proof reading function This ensures a high degree of sequence accuracy The amplified cDNA is ready for RT PCR studies with no purification necessary Page 2 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A
7. sufficient template for the cloning of selected small RNA gene transcripts processed by RNase Ill Amplification of novel microRNAs can be completed in less than 1 day with 0 5 ug of total RNA B 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 1616 North Shoreline Blvd 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 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual V Licensing and Warranty Statement Limited Use License Use of the Small RNA Amplification and Cloning 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
8. 9 SBI System Biosciences Global MicroRNA Amplification Kit Cat RA400A 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 ver 3 060901 contained in this user manual Small RNA Amplification and Cloning Kit Cat RA400A 1 Contents l Introduction and Background As 3OVerVIeW oon tesoros ote copied rhe eere os teed 2 B MicroRNA and Other Small RNAs _ _ h kone 2 C Small RNA Amplification euius 3 D Overview of Protocol i aa 6 E ListofComponents sss 7 F Additional Required Materials e 7 G Procedural Guidelines eee 8 ll Protocol Ae Starting RNAS o Sot lal by Soe ee Tee ruat Blog 9 B Adaptor Ligation Lusso 9 C First Strand cDNA Synthesis Lusso 10 D Second Strand cDNA Synthesis and Amplification 11 E Gene Specific Amplification Using Amplified cDNA _ _ 12 F Generation of Amplified Sense RNA by T7 IVT 13 lll Troubleshooting A No Product from cDNA Amplification 14 B No Product with Gene Specific Primers sss 14 IV Appendix A Related Products sss 15 B Technical Support sss 15 V Licensing and Warranty Statement 16 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A Overvie
9. array experiments Note that the sizes of the microRNA amplified species including the 60 nt adaptors would range from 79 to 84 nts 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual An example of the representation of small noncoding RNAs following RNA amplification by RT PCR is shown in Figure 2 below microRNAs Ss 2 Sg 8 9 a g S no o eo D 3 QN o A o 1 2 E N co Fig 2 High representation of small RNAs after amplification Small RNA was amplified from 50 ng of a pool of RNAs from 4 different tissues with the Small RNA Amplification and Cloning Kit The amplified cDNA was then tested for the presence of 12 small RNAs including 10 specific miRNAs by RT PCR The number of PCR cycles was adjusted to provide similar amounts of PCR products Differences in cycle number between the different small RNAs were as high as 6 cycles For any method of gene transcript amplification method it is important that the relative levels of RNAs be maintained during the amplification process A quantitation comparison of the levels of small RNA before and after amplification is shown in Fig 3 1000 900 E Non amplified 800 El Amplified ae r r r NET r r Io 22 Q En eo e eo e band intensity a S B o e e e e N o eo e o 3p o Ire x 148b let7a 124a
10. atively stable and can be stored for a few hours at room temperature or 4 C For longer storage place at 20 C e Itis important to perform the amplification with the Control RNA provided with this kit Without this control reaction it will be difficult to troubleshoot any unexpected results 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual ll Protocol A Starting RNA We recommend starting with approximately 100 ng of total RNA Our studies have consistently shown greater than 95 representation of RNA species in the amplified population when compared with unamplified RNA with starting concentrations greater than 100 ng The minimum starting concentration we recommend is 5 ng of total RNA IMPORTANT NOTE The RNA isolated by the investigator must contain small RNA We suggest using RNA isolated using an acid phenol method of extraction such as TRIzol Invitrogen Cat 15596 026 We recommend Ambion s mirVana miRNA Isolation Kit Cat 1560 for the isolation of small RNAs B Adaptor Ligation 1 For each RNA sample add the components to a 0 2 or 0 5 ml PCR tube in the order specified 2 0 ul RNase free Water 5 0 pl Ligase buffer warmed to 37 C before use VERY IMPORTANT 0 5 ul 3 Adaptor 2 0 ul Total RNA 0 5 ul Ligase Cocktail 10 0 ul Total volume Note Because reagent volumes are small accurate pipetting is critical 2 Incubate the reactions at 37
11. evealing the role miRNAs play in cellular development and disease SBI has developed a simple and reliable method for preamplification of small RNAs for either qRT PCR or miRNA microarray studies There are estimated to be hundreds of distinct miRNAs in mammalian cells not including the many other small noncoding RNAs Because it is clear that a specific miRNA can influence the gene expression level of multiple genes it is important to define the cell and tissue specificity as well as the spatial and temporal expression pattern of miRNAs However the detection of miRNA expression has been met with significant difficulties mostly due to their small size and low abundance Originally levels of miRNA were examined by Northern blot assays However this requires very large amounts of RNA typically 10 15 ug Recently the highly sensitive method of real time quantitative PCR and highly scalable gene expression microarrays often used to study the expression patterns of conventional mRNAs have been applied to the study of miRNAs These two techniques have advantages and disadvantages Real time qPCR is highly sensitive but has low throughput and miRNA microarray studies have lower sensitivity but are highly scalable Since there are probably 2 500 distinct miRNAs miRNA microarrays provide an effective primary screening method However miRNA microarrays require a large amount of starting total RNA with several publications having described the use
12. ions or concerns about any SBI products please contact us at 888 266 5066 2007 System Biosciences SBI Page 14 ver 5 072612 www systembio com
13. lar RNA sample more cycles may be necessary If so perform an additional 2 cycles and check your amplified cDNA again You do not need to add additional PCR Polymerase even if your reaction was cycled overnight as long as you held the reaction at 15 C after cycling You can continue adding two cycle increments until you see the sufficient product from your amplification reaction however you should not exceed 50 cycles The amplified cDNA may be stored at 4 C for a couple of weeks For long term storage we recommend storing the cDNA at 20 C amplified cDNA bp 500 400 200 400 region of 50 mature microRNA together with adaptor Fig 5 Amplification Results As shown when 2 5 ul of the amplified reaction with the Control RNA was run on a 3 agarose gel the amplification process should generate a smear of cDNA with a size range from 150 bp to approximately 1 500 bp The smear will look a little different depending on whether your starting RNA was mostly intact or somewhat degraded For intact starting RNA the average size of the amplified cDNA should be approximately 500 bp If you do not see this smear refer to the Troubleshooting Section of this manual M 100 bp DNA Ladder 50 bp 2 000 bp E Gene Specific Amplification Using Amplified cDNA The amplified cDNA is ready to use as a template without purification However you must denature the amplified cDNA by incubating for 10 minutes at 95 C before starting gene s
14. n specific product with your gene specific primers may be a problem with your PCR reagents Try to amplify with primers specific for an abundant gene such as mi16 or mi24 which are ubiquitously expressed miRNA If the PCR still fails to generate a product after 35 cycles try using new PCR reagents and enzyme It is critical that you use a non hotstart PCR polymerase We typically use Ambion s SuperTaq Polymerase Cat 2052 Page 12 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A 1 IV Appendix A Related Products e Full Spectrum Global mRNA Amplification Kit Cat RA101A 1 The Full Spectrum Global mRNA Amplification Kit provides an inexpensive method to amplify reverse transcribed RNA in a sequence independent unbiased and uniform manner This approach maintains the relative levels of each transcript in the starting mRNA samples even when using starting amounts of RNA as low as 5 ng or when using heavily degraded RNA e Full Spectrum MultiStart Primers for T7 IVT Cat RA300A 2 Extract more data from RNA than currently available primers in nearly all commercially available T7 IVT kits using Full Spectrum technology Just replace the existing T7 primer with the Full Spectrum primers Compatible with Affymetrix GeneChip hybridization e MicroRNA Discovery Kit Cat RA410A 1 Uniformly and reproducibly amplify limited amounts of non degraded as well as degraded RNA to provide
15. nse RNAs were then tested for the presence of 5 small non coding RNAs including 3 microRNAs 181a 24 and 150 by RT PCR All 5 small RNAs were present in the amplified RNA 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual lll Troubleshooting A No Product from cDNA Amplification If after step D 3 you do not see a smear on the 2 5 3 5 gel for your RNA samples try the following suggestions If you do not see a smear for any samples including the Control RNA One or more of the reagents were omitted during the procedure or the volume of the reactions is incorrect Calibrate your pipette and try amplifying the Control RNA again If you see a smear for the Control RNA but not for your RNA samples You may have less starting RNA than measured Place the amplification reactions back in the thermocycler for an additional three cycles If the expected smear described in Section D 3 is generated you should continue with qRT PCR If after additional cycles there is still no smear or a very weak smear compared with the Control RNA reaction your RNA may either be 1 too degraded or 2 contain an inhibitor Try the amplification again after repurifying the RNA If you still do not get sufficient yield try a different RNA purification kit No Product with Gene Specific Primers If you have confirmed that the amplification in step D 3 was successful but you do not get product or get no
16. pecific PCR It is critical that you use a non hotstart PCR polymerase We typically use Ambion s SuperTaq Polymerase Cat 2052 We recommend that the amplified cDNA be added as a 1 component of your gene specific PCR reactions Thus for a 25 ul gene specific PCR reaction you should add 0 25 ul of the amplified cDNA for a 50 ul PCR reaction add 0 5 ul of the amplified cDNA etc As small RNA controls we recommend using Ambion s RT PCR primer pairs for 5S rRNA and U6 small RNA Page 10 ver 5 072612 www systembio com Small RNA Amplification and Cloning Kit Cat RA400A 1 F Generation of Amplified Sense RNA by T7 IVT To generate labeled hybridization targets for microRNA microarrays the amplified cDNA can be used as template for T7 based in vitro transcription T7 IVT in the presence of fluorescent labeled nucleotides by standard methods An example of a T7 IVT experiment is shown in Figures 6A and 6B 100 to 250 ng of amplified and purified DNA will yield between 10 25 ug of amplified RNA sense M RNA bp 500 400 200 100 50 Fig 6A 50 ng of a pool of RNAs from 4 different tissues was amplified with the SBI kit and purified A small sample of the amplified cDNA was subjected to T7 IVT and then electrophoresed on a 3 agarose gel The yield of amplified RNA was 22 ug M 5S UG 181a 24 150 M Fig 6B A small sample of the amplified sense RNA from Figure 6A was used as a template for T7 IVT The amplified se
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18. w This manual provides details and information necessary to use the Small RNA Amplification and Cloning Kit previously known as the Global MicroRNA Amplification Kit to uniformly and reproducibly amplify limited amounts of non degraded as well as degraded RNA to provide sufficient template for quantitative PCR of selected small RNA gene transcripts To ensure optimal results please read the entire manual before using the reagents and material supplied with this kit B MicroRNA and Other Small RNAs The study of noncoding RNAs especially noncoding micro RNAs miRNA has gained increasing attention in recent years Micro RNAs are 19 24 nucleotide long single stranded RNAs that regulate the expression of target genes by interacting with complementary sites in the 3 UTR of the target mRNAs and inhibiting translation MiRNAs are a conserved group of noncoding RNAs with very important regulatory roles Mature miRNAs are excised from stem loop precursors which are themselves transcribed as part of longer primary transcripts These primary miRNAs appear to be processed by the RNAse Drosha in the nucleus and the precursor miRNAs are exported to the cytoplasm where they are further processed by an RNAse Dicer These enzymes are known to be important for the generation of mature small inhibitory RNAs of exogenously transferred small inhibitory RNA precursors Determining the level of miRNAs has recently become a significant step in the progression of r
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