Sharpvue™ miRNA First Strand Kit
Contents
1. Preparation Wearing a lab coat disposable gloves and protective goggles are recommended when handling chemicals RNA Sample Preparation When working with RNA it is important to avoid RNases in your solutions consumables and labware When preparing your RNA samples always wear a mask and disposable gloves in all procedures Follow the described procedures you are using for RNA extraction carefully Ready to use solutions that are RNase free can be purchased Alternatively treat solutions with diethyl pyrocarbonate DEPC and then autoclave RNases on labware can also be inactivated by DEPC treatment or by baking at 250 C for 3 hours Use DEPC to treat all microcentrifuge tubes pipettes tips if not RNase free and then autoclave to deactivate RNase RNase free consumable are available for purchase from many commercial sources IMPORTANT NOTES 1 Store kit at 20 C Avoid storage or leaving reagents at 4 C or room temperature 2 Touch gently to avoid shocking severely then briefly centrifugr before use 3 Set up all reactions on ice to reduce risk of RNA degradation 4 Read all procedures before setting up RT reaction Procedure 1 Poly A Tailing and Reverse Transcription a Set the following components on ice Add the following reagents into an RNase free reaction tube which has been pre cooled on ice The final volume should be 101 Component Volume ul Total RNA X Sharpvue miRNA First Strand Kit 5x Mi2. PCR by using forward primer reverse primer and 2X Sharpvue qPCR Master Mix Cat No 9000007 Proprietary primer design and its combination with the use of Sharpvue qPCR Master Mix assure extraordinary sensitivity specificity and equal efficiency Sharpvue qPCR Master Mix uses our proprietary DNA polymerase microTaq This enzyme remains inactive in the master mix until after 2 minutes at 95 C The active form of microTaq is so efficient in amplifying small amplicons that LNA containing primers are not needed Related Products Sharpvue miRNA Assay Product Name Description High sensitivity and specificity easy to operate Non toxic EvaGreen based real time quantitative PCR Mix miRNA RT Kit Sharpvue 2x Universal qPCR Master Mix High Rox Human miRNA Assay Primer Sets covering 1700 human miRNAs from latest Human miRNA Primer Array Set v1 0 384 well miRBase release of v17 0 two forms of Human miRNA Primer Array Set v1 0 96 well design are 5 and 20 plates Sharpvue Gene Expression Assay Gene First Strand Kit Accurate quantification of mRNA expression Non toxicEvaGreen based real time quantitative PCR Mix Sharpvue 2x Universal qPCR Master Mix High Rox Contents and Storage Contents Quantity Storage temperature conditions 5x MixA 25 reacction 20 C 15xMixB 25 reacction 20 C dd H20 RNase and DNase free Iml Room temperature
3. _ Biovue Technology Sharpvue miRNA First Strand Kit For reliable first strand cDNA synthesis from all miRNA sources Cat No 9000004 25 reactions User Manual I Sharpvue miRNA First Strand Kit Sharpvue miRNA Assay is the lasted product from Biovue It is an EvaGreen dye based real time qPCR method for specific and quantitative detection of mirco RNA Description Comparing with similar products from other international manufacturers on the market Biovue boasts 1 the largest collection of validated Assays gt 1700 human miRNAs in miRBase v 17 0 2 one of the largest detection range up to 6 magnitude as shown in Figure 1 3 the most sensitive detection down to a single copy number as shown in Figure 1 4 the highest specificity less than 3 cross reactivity among 8 members of let7 family which is almost half of the second best performer in the market today and 5 equal or almost equal efficiency in amplification of each individual miRNA This assay starts with total RNA which must includes miRNA Customers may get purified total RNA with a kit from Qiagen or other manufacturers In the first step by using Sharpvue miRNA First Strand Kit poly A tails are added to the whole population of miRNA and immediately the tailed miRNA population is converted into a population of cDNA from the RT primer Next a particular species of derived cDNA is quantified and differentiated from the rest in the next q
4. duct for a particular purpose Biovue is committed to providing our customers with high quality products If you should have any questions or concerns about any Biovue products please contact us at 86 21 34612632 Biovue Technology Shanghai China Ltd 2nd Floor 1st Building 2140 XieTu Road Shanghai 200032 China www biovuetech com Tel 86 21 34612632 Fax 86 21 64183997 Email order biovuetech com service biovuetech com support biovuetech com
5. is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products without prior written consent from Biovue This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty Biovue warrants that the Product meets the specification described in the accompanying Product Datasheet If it is proven to the satisfaction of Biovue that the Product fails to meet these specifications Biovue will replace the Product In the event a replacement cannot be provided Biovue will 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 Biovue 30 days of receipt of the Product Biovue s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price Biovue s liabilitydoes 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 Biovue does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Pro
6. product RNA template degradation The quality of RNA is the key factor for cDNA synthesis Follow the RNA isolation kit procedure carefully always wearing a lab coat gloves and maskwhen working with RNA and use RNA Grade reagents and materials Check the RNA quality by RNA electrophoresis in a denaturing gel An inhibitor was present in the RNA template Trace amounts of inhibitor such as guanidine salts in the RNA template can inhibit the cDNA synthesis Re precipitate the RNA with ethanol and wash the pellet with 75 ethanol A G C rich template or secondary structure of the amplification product is obstructing the reaction Prepare the RNA Primer Mix before the RT step Then add a PCR enhancing reagent such as DMSO betaine etc in the PCR reaction PCR product is longer than expected Genomic DNA was present Perform a DNase I digest before the RT step or design intron spanning or flanking primers to avoid co amplification of genomic DNA The wrong product was amplified Optimize the PCR reaction conditions Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of all miRNAs and Packaging Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to Biovue within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product
7. r e g ABI 7900 d Set the thermal profile as follows Cycling Step Temperature HoldingTime Number of Cycles Enzyme hot activation 96 C 2 minutes 1 Denaturation 96 C 5 seconds 40 Annealing 60 C 30 seconds 96 C 15 seconds Melting analysis 60 C 15 seconds 1 96 C 15 seconds Set the optical parameters as follows Reporter SYBR or FAM Passive reference for some instrument ROX Example The amplification efficiency and detection sensitivity of the Sharpvue miRNA First Strand Kit are assessed by standard curves made by gradient dilution of the miRNA let 7a Dissociation Curve Amplification Plot Amplification Plot 1 000 E 260 61 2 100 61 10061 1 800 61 ARn 1 100 61 1 000 E2 6 000 62 1000 63 4 1000 62 1000 644 Cycle H Detector STER Piot ARn vs Cycle x color Wen z ee wm 750 wo Temperature C T Anis Log Threshold 0 20 Detector SUER v Plot Derivative v Step Stage 3 step 3 Amplification curves of serially diluted miRNA 7a Peak values of amplified products in melting curves The peak values from the amplification and melting curves show that as low as 0 001 pM can be detected when using miRNA 7a as a template and that there is only a single amplified product showing that very high sensitivity can be attained using the Sharpvue miRNA First Strand Kit Trouble Shooting Guide Little for no RT PCR
8. x A Cat No 9000005 2 Sharpvue miRNA First Strand Kit 15x Mix B Cat No 9000006 0 67 Nuclease free HO Cat No 9000016 to 10 Total 10 For Multiple reactions prepare a master mix of common components b Mix gently and spin the tube briefly to collect the contents c Transfer the tubes to a thermal cycler Incubate at 37 C for 60 minutes d Inactivate the reaction at 85 Cfor 5 minutes e Store the single stranded cDNA at 20 C or proceed directly to PCR amplification 2 qPCR a Set the following components Component Volume ul Forward Reverse miRNA primer set each 3 33x 3 00 RT product from step 1 0 67 2X Shanrvue qPCR Master Mix High Rox Cat No 9000007 5 00 Nuclease Free Water Cat No 9000016 1 33 Total 10 00 Component Volume ul Forward Reverse miRNA primer Array 384 well plate 0 RT product from step 1 0 67 2X Shanrvue qPCR Master Mix High Rox Cat No 9000007 5 00 Nuclease Free Water Cat No 9000016 4 33 Total 10 00 Component Volume ul Forward Reverse miRNA primer Array 96 well plate 0 RT product from step 1 0 67 2X Shanrvue qPCR Master Mix High Rox Cat No 9000007 5 00 Nuclease Free Water Cat No 9000016 4 33 Total 10 00 b Seal the plates with qPCR film and mix gently then spin the tube briefly to collect the contents c Transfer the plate to a real time thermal cycle
Download Pdf Manuals
Related Search