Product Name : Simple mirna Detection Kit

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1 Product Name : Simple mirna Detection Kit Code No. : DS700 This product is for research use only Kit Contents This kit provides sufficient reagents to perform 20 reactions for detecting microrna. Components Amount Storage 1 2 Ligation Buffer 100 µl - 20 C 2 RNase-free water 1 ml - 20 C 3 Ligase 3 20 µl - 20 C 4 Optimum RT Buffer 180 µl - 20 C 5 Reverse Transcriptase 20 µl - 20 C 6 2 Ampli Buffer 500 µl - 20 C 7 Taq DNA polymerase 40 µl - 20 C Store at -20 C (Do not store in a frost-free freezer) The product is stable under the above storage conditions for 2 years from the date of receipt. Additional Materials and Equipments Needed MicroRNA specific primer (Target RNA specific primer) PCR tubes or 96-well plates Thermal cycler Polyacrylamide gel (10%) Apparatus for polyacrylamide gel electrophoresis Power supply Microcentrifuge Ice bucket Vortex mixer - 1 -

2 Experiment Outline The Simple mirna Detection Kit is a product to detect microrna. The detection system is composed of three steps. In the first step, microrna in the solution is directly ligated to an oligonucleotide tag (ON Tag). The tagged microrna is reverse-transcribed in the second step. In the final step, the resulting cdna is amplified with a microrna specific primer of the user s choice. We recommend examining the PCR products with polyacrylamide gel electrophoresis but not agarose gel electrophoresis for high sensitivity and accuracy. The ON Tag can be ligated to 3 -OH of microrna, even if the 2 -OH at the 3 end of the microrna is methylated (1). The microrna specific primers can be prepared based on the 5 sequence of the target microrna. This gives you flexibility to detect microrna from many sources. The Simple mirna Detection Kit can be utilized not only for microrna detection but also the detection of RNA bearing a 3 -OH end which is similar in size to microrna. Fig. 1 MicroRNA detection scheme 1. ON Tag Ligation to MicroRNA 5 P Ligation OH 3 5 ON Tag 2. Reverse-Transcription Reaction Reverse transcription RT Primer 3. PCR Amplification microrna specific primer Universal Reverse Primer Amplified product - 2 -

3 Before Starting 1. Design a microrna specific primer To detect microrna of your interest, a microrna specific primer (forward sense primer) is required to be prepared. A reverse primer (antisense primer) is provided as a Universal Reverse Primer which is included in the 2 Ampli Buffer. Generally micrornas range in size from nucleotides. The seed sequence is at positions 2-7 from the 5 -end of microrna. The sequence is essential for the binding to the mrna and conserved (2). In most cases, the microrna specific primer can be designed directly according to nucleotides of sequence on the 5' side of the microrna. The Tm* of the primer is best to be C. Self-complementary or complementary to the Universal Reverse Primer should be avoided. In some cases, primer dimers may disturb the microrna detection. To avoid this, we recommend that the microrna specific primer is tested by this detection system without RNA. To be more definite, perform ON Tag ligation using pure water instead of RNA, and subject the ligated product to a reverse-transcription reaction, then test whether the microrna specific primer causes bands or not on PCR amplification according to this protocol (See Troubleshooting ). If bands are seen, the microrna specific primer is not appropriate. The microrna specific primer should be prepared in RNase-free water and the concentration should be adjusted to 3 µm. *The equation for Tm: 4(G + C) + 2(A + T) C. A, G, C, and T are the number of each nucleotide in a primer. The sequence of Universal Reveres Primer: 5 GGTGCATTGATTCCCGAGT (19 mer) An example of microrna specific primer design for hsa-mir-93 is shown below: Sequence of hsa-mir-93: CAAAGUGCUGUUCGUGCAGGUAG Primer 1: CAAAGTGCTGTTCGTGCAG (19 nt) Primer 2: AAAGTGCTGTTCGTGCAGG (19 nt) bp Fig 1. Electrophoresis of PCR products PCR products obtained with the Simple mirna Detection Kit were run on 10% polyacrylamide with 1 TBE as running buffer. Visualized by ethidium bromide staining. Lane 1: DynaMarker Small DNA Lane 2: PCR product with Primer 1 Lane 3: PCR product with Primer 2 Both of the primer 1 & 2 work successfully as shown in the Fig. In the above example, the size of the amplicon from hsa-mir-93 as a template can be calculated as below: The amplicon with Primer 1: 47 bp = 23 (length of hsa-mir-93) + 24 *1 The amplicon with Primer 2: 46 bp = 23 (length of hsa-mir-93) -1 * *1 *1 : the nucleotide length formed with Universal Reverse Primer *2 : See the position of Primer 2 on the sequence of hsa-mir

4 2. Preparation of polyacrylamide gel Non-denaturing polyacrylamide gel electrophoresis is appropriate for analysis of the PCR products yielded in the Simple mirna Detection Kit. The electrophoresis can show the PCR band clearly and indicate the size accurately. Preparation of non-denaturing polyacrylamide gel (10%) is described in the section Procedure for Detection below. If the prepared gel plate is wrapped in film, the gel plate can be stored for at least one week at room temperature. 3. Notes on handling of RNA RNA is a labile molecule and easily degraded by RNase. To avoid RNases, use extreme care during manipulation to prevent nuclease contamination. Wear gloves and use clean apparatus. Nuclease-free disposable plasticware should be used. It is necessary to ensure that total RNA or fractionated RNA for microrna detection preserves small RNAs. You can use total RNA prepared by guanidinium thiocyanate-phenol-chloroform extraction method (3) or by commercial total RNA preparation kits. When using a commercial kit, be sure that the kit does not deplete small RNAs. Procedure for Detection In this procedure below, 50 pg to 1 µg of total RNA can be used as a starting sample. For low-content micrornas in the total RNA or in the fractionated RNA (for example, size-selected RNA), an increased amount may be required. The proper amount of RNA depends on the abundance of the microrna target in the sample. 1. ON Tag ligation to microrna Set up the following 10 µl reaction in a 0.5 ml nuclease-free tube and incubate at room temperature (20-25 C) for 15 min. After the 15 min, the ligation mixture is subjected to a reverse-transcription reaction in the next step. Table 1. Reaction mixture for ON Tag ligation to mirna Component Volume RNA (total RNA, fractionated RNA) variable 2 Ligation Buffer 5.0 µl RNase-free water variable Ligase µl ligation mixture 10.0 µl 2 Ligation Buffer includes ON Tag

5 2. Reverse-transcription Reaction 1) Add the following components below to the ligation mixture obtained in the above reaction, and incubate at 42 C for 30 min. Table 2. Reaction mixture for reverse-transcription reaction Component Volume Ligation mixture (see above) 10.0 µl Optimum RT Buffer 9.0 µl Reverse Transcriptase 1.0 µl RT product 20.0 µl Optimum RT Buffer includes RT primer. 2) Terminate the reaction by heating at 95 C for 5 min (This step is important). * The RT product can be stored at -80 C. Repeated freeze-thaw should be avoided. 3. PCR with microrna specific primer 1) Using the 4 µl of RT product prepared above, prepare a reaction mixture as below in a tube or a plate. Table 3. Reaction mixture for PCR Component Volume RT product (see above) 4.0 µl RNase-free water 16.5 µl 2 Ampli Buffer 25.0 µl 3 μm microrna specific primer 2.5 µl Taq DNA polymerase 2.0 µl Total volume 50.0 µl 2 Ampli Buffer includes Universal Reverse Primer and provides a final concentration of 2.8 mm MgCl 2. 2) Transfer the tube or the plate to a thermal cycler* then start PCR according to the PCR program below. * When using a thermal cycler without a heated lid, overlay with mineral oil. Initial denaturation 95 C for 3 min 30 Cycling 94 C for 15 sec 56 C for 30 sec 72 C for 30 sec Final Extension 72 C for 1 min 3. Examine the amplified products The amplified products obtained in the above reaction will be analyzed with non-denaturing polyacrylamide gel electrophoresis as below. 1) Preparation of 40% acrylamide : bis solution Dissolve acrylamide and N, N -methylenebisacrylamide as follows: Acrylamide 190 g N, N -methylenebisacrylamide 10 g H 2 O to 500 ml After mixing, filter the solution through a membrane filter (0.45 μm pore size)

6 2) Preparation of polyacrylamide gel plate To analyze the amplified products of the Simple mirna Detection Kit, 10% polyacrylamide is appropriate. Prepare 10% acrylamide solution as below and assemble glass plates for electrophoresis in a casting frame: 40% acrylamide : bis solution 5.0 ml 10 TBE 2.0 ml H 2 O to 20 ml To the 20 ml solution, add 20 μl of TEMED and 160 μl of 10% ammonium persulfate. Mix immediately and then pour the gel between the glass plates in a casting frame. Then quickly insert the appropriate comb between the glass plates. The 20 ml of gel solution is enough for the two gel plates described in Fig 2 (gel size: 7 cm 8 cm, 1.0 mm thickness, lane width = 5 mm). Be careful not to allow air under the comb. Let the gel sit until it is solid. 0.5 cm comb (1.0 mm thickness) 7 cm 10% polyacrylamide gel (between glass plates) 8 cm Fig.2. Example of a polyacrylamide gel plate 3) Electrophoresis Prepare loading samples by mixing amplified products of the Simple mirna Detection Kit and gel loading buffer (for example, 6 BPB Loading Dye, BioDynamics Laboratory, Code. No. DM210). Set up the gel apparatus according to the manufacturer s protocol. Before electrophoresis, remove the comb from the polyacrylamide gel plate and install it in the electrophoresis apparatus. Fill the apparatus with 1 TBE. Load the samples into wells with a pipet using gel loading tips. Start the electrophoresis according to the manufacturer s protocol. 4) Staining After electrophoresis, detach the gel from the glass plates and immerse the gel in a solution containing 0.5 μg/ml of ethidium bromide approximately for 20 min. Then examine the gel on a UV illuminator

7 Troubleshooting Problem No bands (or faint bands) were seen in the electrophoresed gel Unspecific bands are seen in the electrophoresed gel The size of amplified band is different from that of expected. Probable Cause & Suggested Solution Starting RNA is low. Even though the protocol is designed to detect microrna in as little as 50 pg of total RNA, actual microrna content depends on the expression level and RNA source. In this case, try to increase the amount of RNA if the samples are not limited. The microrna specific primer may not anneal well to a template. In this case, the best solution is to change the microrna specific primer. Longer primers (20, 21 mer) may work well. Alternatively, lowering the annealing temperature of the PCR cycle may improve the PCR amplification. If the microrna specific primer is self-complementary or complementary to the Universal Reverse Primer, unspecific bands such as primer dimers, oligomers resulting from primer dimers may be produced. In this case, the microrna specific primer should be changed. Although primer dimer is a low probability event in using the Simple mirna Detection Kit, primer dimers may be generated in some cases. To avoid it, the designed microrna specific primer should be tested as follows: Perform the ON Tag ligation using pure water instead of RNA, and subject the 10 µl of ligated product to reverse-transcription reaction, then apply 4 µl of the reverse transcribed product to PCR according to this protocol. If you see bands other than primers, they must be primer dimers or oligomers resulting from primer dimers. In this case, the microrna specific primer should be changed. See Fig. 3 The PCR over 30 cycles sometimes causes unexpected bands. In this case, perform PCR in less than 30 cycles. Reverse-transcribed cdna can be stored below -20 C but freeze-thaw cycle may cause unexpected bands. bp Fig 3. A generated primer dimer ON Tag ligation was done with human heart total RNA (lane 3) and without RNA (lane 2), then the ligated products were subjected to a reverse-transcription reaction. PCR was done with the reverse-transcribed products and an inappropriate microrna specific primer for PCR. Approximately 42-bp bands in lane 2 and 3 are primer dimers. The bands disturb the detection of correct bands. Lane 1: DynaMarker Small DNA Lane 2: PCR product (- total RNA) Lane 3: PCR product (+ total RNA) - 7 -

8 Trademarks Registered names, company names, trademarks, etc. used in this document are the property of their respective owners, even when not specifically marked as such. References 1. Yang Z, Ebright YW, Yu B, Chen X. (2006) HEN1 recognizes nt small RNA duplexes and deposits a methyl group onto the 2 OH of the 3 terminal nucleotide. Nucleic Acids Res 34: Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets. Cell 120: Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:

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