BIOO RESEARCH PRODUCTS

Transcription

1 BIOO RESEARCH PRODUCTS FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit and protocol, so it is important to always use the protocol included with the kit. MiraMas Kit Manual Catalog #: 5208 BIOO Scientific Corp. 2010

2 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Controls Included In The Kit... 1 Procedure Overview... 1 Kit Contents, Storage and Shelf Life... 2 Required Materials/Equipment Not Provided With the Kit... 2 Warnings and Precautions... 3 RNA Sample Requirements... 3 Number of Samples That Can Be Processed... 4 FORWARD PRIMERS FOR MICRO RNA AMPLIFICATION... 4 MIRAMAS PROTOCOLS... 5 Format H for Reactions Containing High Amounts of Input RNA... 5 Format L for Reactions Containing Low Amounts of Input RNA... 7 Running the Positive and Negative Control Reactions APPENDICES Appendix A...11 Appendix B...11 Appendix C RELATED PRODUCTS The MiraMas Kit is intended for laboratory use only. MiraMas is a trademark of Bioo Scientific.

3 General Information GENERAL INFORMATION Product Description MiraMas Kit Manual 5208 The MiraMas Kit is designed to create cdna libraries to use for qpcr detection of micrornas and other small RNAs. The method is based on ligation of a 5 - adenylated/3 blocked oligonucleotide adapter (Adenylated 3 Ligation Adapter) to the 3 ends of the small RNAs using RNA Ligase, followed by reverse transcription with M-MLV Reverse Transcriptase to convert the small RNAs to cdna templates for qpcr. The ligated adapter provides the binding site for the reverse transcription (RT) primer. After the second-strand cdna is synthesized by extension of a microrna-specific Forward PCR primer, the same adapter sequence serves as binding site for the Reverse PCR primer. Use of an adenylated adapter is advantageous because it streamlines the work-flow and leads to increased specificity for producing the desired reaction products, with fewer unwanted side products. The MiraMas protocol uses a single-tube format for ligation, reverse transcription, and subsequent dilution of the cdna library. The MiraMas Kit allows users great flexibility in formatting their experiments, in that the number of samples that can be processed is scaled to the amount of sample RNA used in the reactions. Depending on the amount of sample RNA, the kit can be used to produce cdna libraries from 30 to 150 samples. Each cdna library can be used for detection of tens to hundreds of target micrornas. Controls Included In The Kit The MiraMas Kit contains two control RNA samples and three microrna-specific Forward control primers. The control RNAs are small RNA enriched fractions from mouse liver and mouse brain. The Forward control primers are designed to detect mir-124 (mainly expressed in brain), mir-122 (mainly expressed in liver) and mir-16 (expressed in most tissues). Procedure Overview The first step of the MiraMas procedure is ligation of the Adenylated 3 Ligation Adapter to the 3 ends of a total RNA or small RNA enriched sample, using AIR Ligase, a truncated T4 RNA Ligase 2. Due to the high specificity of the ligation step, no purification of reaction products is required; the reaction is terminated by simply heating to inactivate the ligase. The RT reaction is then carried out in the same tube. The second step of the procedure is reverse transcription of the ligated sample with M-MLV reverse transcriptase (MMLV-RT). This step is primed using the RT Primer, which is complementary to the Adenylated 3 Ligation Adapter. This step creates a cdna library of small RNAs. The RT reaction is then diluted in the same tube for use as template in the qpcr step. The last step of the procedure is to carry out real-time PCR, using the Universal Reverse PCR primer included with the kit along with a user-supplied Forward primer that is specific for the microrna target. The kit includes enough Universal Reverse Primer for 1,000 real-time PCRs. BIOO RESEARCH PRODUCTS 1

4 Kit Contents, Storage and Shelf Life Note, all components except the water should be stored at -20 C in a non-frost-free freezer. The water may be stored at room temp. The Primer Resuspension Solution can be stored at room temp after dilution to 1X. When properly stored, the kit will maintain activity for at least 6 months after receipt. Component Amount supplied in kit Ligation Components: Yellow Caps Adenylated 3 Ligation Adapter One nanomole (lyophilized) Adapter Resuspension Buffer 500 µl AIR Ligase (RNA ligase) 55 µl (200 units/ µl) 10X Ligase Reaction Buffer 200 µl PEG (polyethylene glycol) 350 µl (50% solution) RT Components: Blue Caps 10X RT buffer 650 µl 4 dntp mix 100 µl (10 mm concentration of each dntp) RT primer 165 µl (50 µm concentration) MMLV-RT(reverse transcriptase) 66 µl (200 units/ µl) Water (nuclease-free distilled deionized) 50 ml PCR components: Green Caps Universal Reverse PCR Primer 3 ml (2 µm concentration) 50 X Primer Resuspension Soln 2 X 1 ml (375 mm Tris-Cl ph 8 / 5 mm EDTA) Control Components: Pink Caps Forward Primer for mir µl (2 µm concentration) Forward Primer for mir µl (2 µm concentration) Forward Primer for mir µl (2 µm concentration) Control RNA #1 (Small RNA- enriched RNA 10 µl, 100 ng/ µl (1 µg total) from mouse brain) Control RNA #2 (Small RNA- enriched RNA 10 µl, 100 ng/ µl (1 µg total) from mouse liver) Required Materials/Equipment Not Provided With the Kit Heat block Vortex mixer Real-time thermalcycler 2X SYBR-real-time Master Mix (recommended: SYBR Green JumpStart Taq ReadyMix from Sigma Life Science) microrna-specific Forward primer(s) Pipettors, tips, microfuge tubes RNA sample(s), extracted using method to recover small RNA (see below, RNA Sample Requirements ) BIOO RESEARCH PRODUCTS 2

5 Warnings and Precautions Many components have similar names, read the labels carefully! Wear gloves when handling the kit components Store the kit in non-frost-free freezer (one that does not cycle to higher temperatures) Reverse Transcription reactions (i.e. the cdna libraries produced with the kit) should also be stored in a non-frost-free freezer for long-term storage The enzymes (AIR RNA Ligase and MMLV-RT) are supplied in buffers containing glycerol. Pipet these reagents carefully to avoid waste Briefly centrifuge ( pop-spin ) all reagent tubes before opening; this prevents loss and contamination of reagents Labels on kit components are applied vertically to allow contents of tubes to be more easily visualized; if labels start to peel off, simply press them back onto the tube Bioo Scientififc makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. Bioo Scientific shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. RNA Sample Requirements A wide range of input amounts of RNA can be used with the MiraMas Kit; in general, input amounts of 10 ng 2,000 ng (0.01 µg 2 µg) are recommended, although amounts outside this range are not necessarily incompatible with the kit. Either total RNA or the small RNA enriched fraction of RNA can be used. Using sample enriched for small RNA is recommended, especially for low-abundance targets. For RNA extraction, it is important to use a method that recovers total RNA that includes the small RNA fraction. Many silica filter-based RNA extraction kits do not recover RNAs less than ~ 100 bases. For extraction of total RNA, with the option of separately recovering the small RNA fraction, we recommend using Bioo Scientific s single-phase RNA extraction reagent, BiooPure (cat # 5301). Small RNA enrichment using the BiooPure protocol does not require gel electrophoresis or specialized equipment. The method is based on differential precipitation of larger RNAs (> 100 bases) using a low concentration of isopropanol, followed by precipitation of small RNA from the supernatant at higher concentration of isopropanol. Other single-phase RNA extraction reagents (e.g. TriReagent, Trizol) can also be used for enriching the small RNA fraction by differential precipitation with isopropanol. The BiooPure reagent however offers an advantage over similar reagents, in that it is supplied with an inert co-precipitant (linear polyacrylamide) to maximize recovery of small RNA. For extraction of RNA from cell-free fluids such as serum, plasma, and cell culture media, we recommend Bioo Scientific s ExoMir Kit (cat # 5145), which includes BiooPure and special syringe filters for concentrating exosomes, microvesicles, and other RNA-containing particles from large volumes of fluids (~10 ml 30 ml). BIOO RESEARCH PRODUCTS 3

6 Number of Samples That Can Be Processed The MiraMas Kit allows users great flexibility in formatting their experiments, in that the number of samples that can be processed is scaled to the amount of sample RNA used in the reactions. Using Format H, for Higher amounts of RNA (0.5 µg 2 µg), cdna libraries can be produced from 30 samples. Using the standard cdna dilution, approximately 200 small RNA targets can then be interrogated per library. Using Format L, for Lower input amounts of RNA (10 ng 300 ng), cdna libraries can be produced from up to 150 samples. Using the standard cdna dilution, up to 25 small RNA targets can then be interrogated per library. The reactions can be freely assorted between Format H and Format L. Note: To compare small RNA profiles between samples, use the same format (Format H or Format L) for making the cdna libraries from all the samples. FORWARD FORWARD PRIMERS PRIMERS FOR MICRO FOR MICRO RNA AMPLIFICATION RNA AMPLIFICATION There are four options for obtaining the microrna-specific Forward primers: Use the standard algorithm described in Appendix A to design primers for your micrornas/small RNAs of interest and order them from a third-party vendor. The mir-124, mir-122, and mir-16 Forward control primers included with the kit were designed using the standard algorithm. Customers may also contact Bioo Scientific at info@biooscientific.com for suggested sequences for their target micrornas. The sequences are designed using the standard algorithm. There is no charge for this service. Design your primers using alternative algorithms. Alternative algorithms may be useful in some cases, for example to design primers that can distinguish between closely-related micrornas (see Appendix A). Use Bioo Scientific s custom primer service. MicroRNA/small RNA-specific Forward primers sufficient for 1,000 qpcr reactions (30 µl qpcr rxns) can be purchased from Bioo Scientific. Validation of primers using customer-supplied RNA is also available. Primer sequences designed using this service will be disclosed to customers. To order primers using Bioo Scientific s custom service or discuss primer validation with your RNA, please contact us at info@biooscientific.com. Order primers from Bioo Scientific s MiraMas microrna Forward primer library (see Appendix B) BIOO RESEARCH PRODUCTS 4

7 Miramas Protocols MIRAMAS PROTOCOLS Format H for Reactions Containing High Amounts of Input RNA Format H Ligation Step (use for reactions containing µg of input RNA): Before using the kit for the first time: Resuspend the Adenylated 3 Ligation Adapter in 33 µl of Adapter Resuspension Buffer (the concentration of adapter will be 30 pmoles per µl). Add the buffer to the dried oligo at the bottom of the tube and vortex thoroughly (~10 sec), then pop-spin to collect all liquid at the bottom of the tube. Use 1 µl per reaction for Format H reactions (using higher amounts of input RNA). For Format L reactions (using lower amounts of input RNA), this component is diluted further, as described in the Format L Protocol. Resuspending the Adenylated 3 Ligation Adapter in 33 µl provides sufficient amount sufficient for 30 Format H reactions, with 10% overage to accommodate pipetting error/loss. Store the resuspended oligo at -20 C. 1. Assemble reactions as shown in the table below. If setting up more than one reaction we recommend using a Master Mix (include 10% overage of reagent volumes in Master Mix to allow for pipetting error/loss). Vortex the Master Mix thoroughly (~ 5 sec) to ensure complete dispersal of the PEG, and pop-spin. Add 5 µl Master Mix per reaction and mix thoroughly with 5 µl of sample RNA. Reactions may be set up in 0.5 ml or 1.5 ml tubes; using 1.5 ml tubes will allow reactions to be diluted in the final step without changing tubes. 2. Incubate reactions for 5 min at 45 C. 3. Incubate reactions for 2 hours at room temp. 4. Incubate reactions for 10 min at 65 C to inactivate the RNA Ligase. After incubation centrifuge the tubes for ~ 5 sec to collect all liquid. Store reactions at -20 C or proceed directly to RT step. Format H Ligation Reaction Component Amount per reaction Sample RNA in water or 0.1 mm EDTA, at 5 µl concentration of 100 ng/µl 400 ng/µl (total RNA input per reaction will be 0.5 µg 2 µg) 10X Ligase Reaction Buffer 1 µl PEG 2 µl Adenylated 3 Ligation Adapter 1 µl (30 pmoles/µl) AIR RNA Ligase (200 units/µl) 1 µl Total Volume 10 µl BIOO RESEARCH PRODUCTS 5

8 Format H Reverse Transcription Step: 1. Assemble reactions as shown in table below. If setting up more than one reaction we recommend using a Master Mix (include 10% overage of reagent volumes in Master Mix to allow for pipetting error/loss). Vortex Master Mix thoroughly and pop-spin. Add 30 µl Master Mix per Ligation Reaction (typically into the same tube used for the Ligation Reaction) and mix thoroughly. Format H RT Reaction Component Amount per reaction Ligation Reaction (from above) 10 µl 10X RT Buffer 4 µl 4 dntp Mix (10 mm each) 1 µl RT Primer (50 µm) 4 µl Nuclease-free water 19 µl MMLV-RT (200 units/µl) 2 µl Total Volume 40 µl 2. Incubate reactions for 1 hr at C. 3. Incubate reactions for 5 min at 90 C to inactivate the reverse transcriptase. Centrifuge tubes for ~ 5 sec. 4. Dilute reactions with distilled deionized nuclease-free water for use in PCR step. The dilutions suggested below can be adjusted based on the amount of input RNA and the abundance of the target in the sample. After adding the water, centrifuge the reactions for ~ 5 sec then pop-spin. Standard dilution of RT reactions made from small RNA enriched samples: Add 860 µl of nuclease-free water. Final reaction volume is 900 µl. The reactions may be diluted further to a total volume of 1.8 ml based on Ct values observed in the initial qpcr. Dilution of RT reactions made with total RNA samples (not small RNA enriched): Dilute with 460 µl of nuclease-free water. Final reaction volume is 500 µl. 5. Store reactions at -20 C or proceed directly to qpcr step. BIOO RESEARCH PRODUCTS 6

9 Format L for Reactions Containing Low Amounts of Input RNA Format L Ligation Step (use for reactions using 10 ng 300 ng of input RNA): 1. Adjust the concentration of the Adenylated 3 Ligation Adapter to 6 pmoles/ µl. If the Adenylated 3 Ligation Adapter was originally resuspended in 33 µl (to make a 30 pmoles/µl solution for use with Format H), adjust the concentration to 6 pmoles/µl for use with Format L by making a 5-fold dilution in Adapter Resuspension Buffer. For example, mix 1 µl of 30 pmoles/µl from Format H, with 4 µl Adapter Resuspension Buffer. Scale up as needed for number of reactions. One µl is used per reaction for reactions using lower amounts of input RNA (10 ng 300 ng). Prepare only enough Adenylated 3 Ligation Adapter as needed for one experiment. For convenience, the dilution may be made directly in the Master Mix tube. If using the kit for the first time: Resuspend the Adenylated 3 Ligation Adapter in 33 µl of Adapter Resuspension Buffer, to give a concentration of ~30 pmoles per µl. Add the buffer to the dried oligo at the bottom of the tube and vortex thoroughly (~10 sec), then pop-spin to collect all liquid at the bottom of the tube. 2. Assemble reactions as shown in table below. If setting up more than one reaction we recommend using a Master Mix. Include 10% overage of reagent volumes in Master Mix to allow for pipetting error/loss. Vortex the Master Mix thoroughly (~ 5 sec) and pop-spin to collect liquid at bottom of tube. Add 5 µl Master Mix per reaction and mix thoroughly. Reactions may be set up in 0.5 ml or 1.5 ml tubes. 3. Incubate reactions for 5 min at 45 C. 4. Incubate reactions for 2 hours at room temp. 5. Incubate reactions for 10 min at 65 C to inactivate the ligase. Store at -20 C or proceed directly to RT step. Format L Ligation Reaction Component Amount per reaction Sample RNA in water or 0.1 mm EDTA at 5.7 µl concentration of 2 ng/µl 60 ng/µl (total RNA input per reaction will be ~10 ng 300 ng) 10X Ligase Reaction Buffer 1 µl PEG 2 µl Adenylated 3 Ligation Adapter 1 µl (diluted to 6 pmoles/µl) AIR RNA Ligase (200 units/ µl) 0.3 µl Total Volume 10 µl BIOO RESEARCH PRODUCTS 7

10 Format L Reverse Transcription Step: 1. Assemble reactions as shown in table below. If setting up more than one reaction we recommend using a Master Mix (include 10% overage of reagent volumes in Master Mix is to allow for pipetting error/loss). Vortex Master Mix thoroughly and pop-spin to collect liquid at bottom of tube. Add 30 µl Master Mix per Ligation Reaction (typically into the same tube used for the Ligation Reaction) and mix thoroughly. Format L RT Reaction Component Amount per 40 µl rxn Ligation Reaction (from above) 10 µl 10X RT Buffer 4 µl 4 dntp Mix (10 mm each) 0.5 µl RT Primer (50 µm) 1 µl Nuclease-free water 24.1 µl MMLV-RT (200 units/µl) 0.4 µl Total Volume 40 µl 2. Incubate reactions for 1 hr at C. 3. Incubate reactions for 5 min at 90 C to inactivate the reverse transcriptase. Centrifuge tubes for ~ 5 sec to collect all liquid. 4. Dilute reactions for use in PCR step. The dilution depends on the amount of input RNA and the abundance of the target in the sample. The standard dilutions shown below may be adjusted with these parameters in mind. Standard dilution of RT reactions that have up to 10 ng RNA: Add 140 µl of nuclease-free water. Final reaction volume is 180 µl. Standard dilution of RT reactions that have ng RNA: Add 320 µl of nuclease-free water. Final reaction volume is 360 µl. 5. Centrifuge reactions for ~ 5 sec then pop-spin. Store at -20 C or proceed directly to qpcr. qpcr for microrna detection Reagent preparation for qpcr Step Make 50 ml of 1X Primer Resuspension Solution, by mixing the 1 ml of 50X Primer Resuspension Solution included with the kit, with 49 ml of nuclease-free distilled deionized water (this water is not included with the kit). The 1X Primer Resuspension Solution may be stored at room temperature. The composition of the 1X Primer Resuspension Solution is 7.5 mm Tris ph 8 / 0.1 mm EDTA. BIOO RESEARCH PRODUCTS 8

11 To make a 2 µm working solution of Forward primer, first resuspend the lyophilized oligonucleotide in the same number of microliters of 1X Primer Resuspension Solution as number of nanomoles of oligo, to make a 1 mm stock solution. For example, if the primer was received as 36 nmoles of lyophilized powder, dissolve it in 36 µl of 1X Primer Resuspension Solution. Then make the 2 µm working solution by making a 1:500 dilution of the 1 mm stock. For example, to make 1 ml of 2 µm working solution, mix 2 µl of 1 mm stock with 998 µl of 1X Primer Resuspension Solution. Store at -20 C. qpcr Step 1. Prepare working solutions of [For + Rev] primers by mixing equal volumes of 2 µm Forward Primer (user-supplied) and 2 µm Universal Reverse PCR Primer provided with the kit. Vortex and pop-spin. For each 30 µl PCR, 6 µl of [For + Rev] primer mix will be needed. Reactions are usually run in duplicate or triplicate. 2. Assemble PCRs in 96-well plates or tubes. Standard 30 µl reactions contain 3 components: Component Volume per 30 µl qpcr cdna sample from diluted RT above 9 µl (For no-template negative controls, use water instead. For minus-rt controls, use RNA*) [For + Rev] primer mixture (1 µm each) 6 µl 2X SYBR qpcr Master Mix 15 µl * Use an amount of RNA corresponding to the amount used to generate the cdna used in the reactions. For example, if the Ligation/RT reaction contained 0.5 µg of RNA and was diluted to 900 µl, use 5 ng of RNA in the minus-rt qpcr. For PCRs carried out in 96-well plates: After adding the components and sealing the plate, gently tap each corner of the plate several times against a hard surface to mix components, then spin the plate in a microfuge to collect all liquid at the bottom of the wells. 3. Run protocol as suggested below or modify according to user experience. Standard qpcr cycling protocol for use with Forward primers designed according standard algorithm: Step 1: Hold 2 min at 95 C Step 2: Cycle 40x: 95 C/10 sec 58 C/30 sec ; carry out data acquisition at 58 C Step 3: Carry out Melt-curve analysis: 55 C 90 C, 0.5 C increments, 10 sec dwell time (requires 81 increments) Analyze results using the thermalcycler-integrated software. BIOO RESEARCH PRODUCTS 9

12 Running the Positive and Negative Control Reactions MiraMas Kit Manual Assemble 10 µl Ligation Reaction(s) as described in the protocols above using the Control #1 RNA (small RNA from mouse brain) and/or Control #2 RNA (small RNA from mouse liver) provided with the kit. The Control RNAs are supplied at a concentration of 100 ng/µl, so up to 500 ng can be used in Format H for high RNA input amounts. For Format L, dilute the Control RNA to a final concentration of 2 ng/µl 60 ng/µl, using nuclease-free water. This will allow input amounts ranging from 10 ng 300 ng to be used in Format L. 2. Use the 10 µl Control RNA Ligation Reaction(s) as input for 40 µl Reverse Transcription Reaction(s) to produce cdna from the samples, as described in the protocols. After the RT step, dilute the reactions as appropriate for the amount of input RNA that was used, as described in the protocols. 3. Prepare working [For + Rev] Control Primer mixtures by mixing equal volumes of the mir-16 For Primer, mir-124 For Primer, and/or mir-122 For primer with the Universal Reverse PCR Primer. All primers are provided at 2 µm concentration, so the mixture will contain 1 µm concentration of each primer. For each 30 µl PCR, 6 µl of [For + Rev] primer mixture will be needed. 4. Set up 30 µl qpcr reactions as described above, using 9 µl of diluted cdna, 6 µl of [For + Rev] primer mix, and 15 µl of 2X SYBR qpcr Master Mix per reaction. Run the standard profile described above. You may want to use the primers to amplify cdna from other tissues, as additional controls. The mir-16 primer mix should produce easily detectable signal from most human and mouse tissues. The mir-124 primers should produce Ct values from Control RNA #1 (brain) that are approximately Cts lower than from Control RNA #2 (liver). The mir-122 primers should produce Ct values from Control RNA #2 (liver) that are approximately Cts lower than from Control RNA #1 (brain). The exact Ct values depend on the input amount of RNA, and on other parameters such as the activity of the PCR Master Mix and the efficiency of the thermalcycler. BIOO RESEARCH PRODUCTS 10

13 Appendices APPENDICES Appendix A Standard algorithm for design of microrna-specific Forward primers The MiraMas kit was developed using microrna-specific Forward primers that span the entire mature microrna sequence, and that include a generic 5-base tag sequence on the 5 end. The 5-base tag sequence is TACGA. Other tag sequences may also be used, but should be tested to verify their performance. The Forward primers generally range from bases in length, do not include modified nucleotides, and are purified using standard de-salting. Forward primers designed in this way and used with the Universal Reverse Primer included with the kit, will produce microrna-specific amplicons that are the same size as the product that would be made by nonspecific primer dimer side-product. Production of primer-dimer would be seen as product in the no-template negative control reaction. Product in the no-template control reactions is typically not observed, when using the standard qpcr profile with Forward primers designed using the standard algorithm. MicroRNA sequences can be easily accessed at the Sanger mirbase website ( a continuously updated public database for microrna-related information. The standard Forward primers have the following format, where the first 5 bases correspond to the 5 base 5 tag and the last ~20-22 bases (X s) are the sequence of the mature microrna: 5 - TACGA-XXX XXX XXX XXX XXX XXX XXX X The Forward primer sequences for several small RNAs, designed using the standard algorithm, are shown in Appendix B below. Alternative algorithm for design of microrna-specific Forward primers and associated alternative Universal Reverse primer An example of an alternative algorithm for designing Forward primers, and the associated sequences of the alternative Forward and Reverse primers, is available for download at: Note, using this alternative primer design requires use of an altered qpcr thermal profile as described in the file. BIOO RESEARCH PRODUCTS 11

14 Appendix B Sequences of microrna-specific Forward primers available from Bioo Scientific. These primers were designed using the Standard Algorithm. All sequences are shown in a 5 3 direction. Small RNA (mmu is mouse; hsa is human) Mature small RNA (mirbase) Forward primers Cat # mmu-mir-16 UAGCAGCACGUAAAUAUUGGCG TACGA TAG CAG CAC GTA AAT ATT GGC G mmu-mir-124a UAAGGCACGCGGUGAAUGCC TACGA TAA GGC ACG CGG TGA ATG CC Mus musculus small CTTTTGGAAC TGAAT CTAAG TACGA CTT TTG GAA CTG AAT nucleolar RNA, C/D box TGATTTAACA AAAATTCGTC CTA AGT GAT TTA ACA 70 (Snord70) LOCUS ACTACCACTG AGA NR_ mmu-mir-191 CAACGGAAUCCCAAAAGCAGCUG TACGA CAA CGG AAT CCC AAA AGC AGC TG hsa-mir-337-5p GAACGGCUUCAUACAGGAGUU TACGA GAACGG CTT CAT ACA GGA GTT mmu-let-7a UGAGGUAGUAGGUUGUAUAGUU TACGA TGA GGT AGT AGG TTG TAT AGT T hsa-mir-21 UAGCUUAUCAGACUGAUGUUGA TACGA TAG CTT ATC AGA CTG ATG TTG A hsa-mir-15a UAGCAGCACAUAAUGGUUUGUG TACGA TAG CAG CAC ATA ATG GTT TGT G mmu-mir-1 UGGAAUGUAAAGAAGUAUGUAU GGCACG TGG AAT GTA AAG AAG TAT GTA T* mmu-mir-122 UGGAGUGUGACAAUGGUGUUUG TACGA TGG AGT GTG ACA ATG GTG TTT G mmu-mir-9 UCUUUGGUUAUCUAGCUGUAUGA TACGA TCTTTGGTTATC TAGCTGTATGA mmu-mir-143 UGAGAUGAAGCACUGUAGCUC TACGA TGA GAT GAA GCA CTG TAG CTC hsa-mir-200c UAAUACUGCCGGGUAAUGAUGGA TACGA TAA TAC TGC CGG GTA ATG ATG GA mmu-mir-150 UCUCCCAACCCUUGUACCAGUG TACGA TCT CCC AAC CCT TGT ACC AGT G mmu-mir-222 AGCUACAUCUGGCUACUGGGU TACGA AGC TAC ATC TGG CTA CTG GGT * The 5 tag sequence was altered to increase the Tm of this primer, since the mir-1 sequence is unusually A/U rich. BIOO RESEARCH PRODUCTS 12

15 Appendix C Sequences of oligonucleuotides included in the MiraMas kit 3 blocked 5 Adenylated 3 Ligation Adapter: 5 -rappcactcgggcaccaagga/3ddc/3 RT primer: 5'- GTC CTT GGT GCC CGA GTG -3' Universal Reverse PCR Primer: 5'- GTC CTT GGT GCC CGA GTG -3' Forward PCR primer for mir-124: 5 - TACGA TAA GGC ACG CGG TGA ATG CC-3 Forward PCR primer for mir-122: 5 - TACGA TGG AGT GTG ACA ATG GTG TTT-3 Forward PCR primer for mir-16: 5 - TACGA TAG CAG CAC GTA AAT ATT GGC G-3 BIOO RESEARCH PRODUCTS 13

16 RELATED PRODUCTS RELATED PRODUCTS RNA Isolation and Enriched Small RNAs: Product Catalog Number BiooPure RNA Isolation Reagent 30 ml BiooPure RNA Isolation Reagent 120 ml mir-mouse Enriched Small RNAs see website Fractionation and RNA Isolation from Exosomes : Product Catalog Number ExoMir Kit 5145 Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group info@biooscientific.com techsupport2@biooscientific.com BIOO RESEARCH PRODUCTS 14