All-in-One™ miRNA qRT-PCR reagent kits user manual
Contents
1. C time 6 sec time Yes 30 C 30 sec No The DNA polymerase used in the 2X All in One qPCR Mix is a chemically especially modified hot start enzyme Incubation for 10 minutes at 95 C will sufficiently activate the enzyme Specific properties of a miRNA lead to special properties of the designed primer Therefore the annealing temperature needs to be strictly controlled in order to avoid non specific amplifications For validated miRNA primers purchased from GeneCopoeia please refer to the optimal conditions for the experiment The Oligo dT Adaptor primer for reverse transcription is 53 nucleotides therefore the resulting PCR amplification fragment is about 75bp assuming the sequence of miRNA is about 22 nucleotides which requires at least about 10 seconds extension time From the melting temperature of the products the Tm value is generally determined to be between 75 C 83 C If the melting temperature exceeds this range other assaying methods such as electrophoresis are suggested for the specific properties of the product All in One miRNA qRT PCR Reagent Kits User Manual v The main conditions for the above reactions are for use with the iQ5 qPCR instrument from Bio Rad If a qPCR instrument from another commercial source is used please reference the instrument manual and adjust the extension time and melting curve conditions accordingly VI Examples a Example 1 Specificity assay using the All in One miRN2. contain small molecule RNA t The amount of total RNA can be between 1 ng 5 pg If using purified small molecule RNA the amount can be between 0 1 ng 1 ug Prepare reverse transcription reaction Mix the prepared reaction mix gently but thoroughly Incubate at 37 C for 60 minutes after a brief centrifugation Incubate at 85 C for 5 minutes to inactivate the enzyme The resulting reverse transcription reaction product should be diluted 5 50 times with sterile H20 before using for the next GPCR experiment or it can be directly stored at 20 C Detection of miRNA with qPCR Dissolve 2X All in One qPCR Mix by gently inverting Briefly centrifuge and place on ice If required dissolve 50X ROX Reference Dye Dilute the 50 uM Universal Adaptor PCR Primer to 2uM with sterile ddH20 before using for the next qPCR experiment Prepare qRT PCR solution on ice See example Reagent Volume Final concentration 2X All in One qPCR Mix 10 ul 1X All in One miRNA qPCR Primer 2 uM 2 ul 0 2 uM Universal Adaptor PCR Primer 2 uM 2 ul 0 2 uM First strand cDNA diluted 1 5 2 ul 50X ROX Reference Dye 0 4 ul 1X Water double distilled a Not using ROX Reference Dye 4ul m Using ROX Reference Dye 3 6 ul Final volume 20 ul All in O ne miRNA qRT PCR Reagent Kits User Manual Notes Use the 2X All in One qPCR Mix as half of the total reaction volume and adjust other reagents according
3. experiment Abnormal melting curves Signals in blank No Template Control sample There may be contamination or positive samples in the PCR reaction system if the Tm of the melting curves of the blank control is the same as the positive control Eliminate sample application error first If the situation still persists change PCR grade water primers or use new 2X All in One g PCR Mix If the Tm of melting curves of blank control is lower than the positive control the PCR reaction may have produced nonspecific amplification such as primer dimers Please prepare PCR reaction mix on ice and increase the temperature of fluorescence detection If the C value of the negative control is gt 35 and the difference with the positive samples is more than 5 cycles the PCR reaction system is up to the standard On the other hand if the ct value cannot reach the aforementioned value then All in O ne miRNA qRT PCR Reagent Kits User Manual redesign the primer or optimize the reaction conditions Double peaks and multiple peaks in melting curves of positive control In the absence of other primers present in the reaction double or multiple peaks in the positive control means that the PCR reaction produces nonspecific amplification fragments Prepare the PCR reaction mix on ice optimize the PCR reaction conditions such as by increasing the annealing temperature decreasing the primer concentration or increasing the fluor
4. A qRT PCR Detection Kit With 200 ng total RNA mixture from human brain and heart as template the miRNA qRT PCR Detection Kit and the All in One miRNA qPCR Primers were used to detect 30 miRNA and an internal reference snRNA U6 Results from qRT PCR and electrophoresis showed neither non specific amplification products nor primer dimer formation r d RFUYT PUR Base Line Subtracted Curve Fit RFU Figure 2 Amplification and melting curves of 31 miRNA and the internal reference snRNA U6 in which double channel detection was used for the positive control and single channel detection was used for the NTC No Template Control Figure 3 Agarose gel electrophoresis 3 agarose gel of the amplification products of 31 miRNA and the internal reference snRNA U6 in which double channel detection was used for the positive control and single channel detection was used for the NTC No Template Control b Example 2 Sensitivity assay using the All in One miRNA qRT PCR Detection Kit Starting with different amounts 5ug 1g 200ng 20ng 2ng 100pg of human brain total RNA the All in One miRNA qRT PCR Detection Kit was used to detect the expression level of hsa miR 124 The results showed that linear amplification can be detected between 5ug 100pg of total RNA All in One miRNA qRT PCR Rea
5. Expressway to Discovery All in One miRNA qRT PCR Reagent Kits For quantitative detection of mature miRNA All in One miRNA First Strand cDNA Synthesis Kit AMRT 0020 20 RT reactions AMRT 0060 60 RT reactions Used in combination with miProfile miRNA qPCR Arrays and All In One qPCR Mix All in One miRNA qRT PCR Detection Kit AOMD Q020 20 RT and 200 qPCR reactions AOMD Q060 60 RT and 600 qPCR reactions Used in combination with All In One miRNA qPCR Primers User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2013 GeneCopoeia Inc All in O ne miRNA qRT PCR Reagent Kits User Manual USER MANUAL All in One miRNA qRT PCR Reagent Kits l Introduction and Principle Il Related Products lll Contents and Storage IV Preparation V Procedure VI Examples VII Trouble Shooting Guide VIII Limited Use License and Warranty I Introduction and Principle Small non coding miRNA are widely present in eukaryotes They consist of about 22 nucleotides that control many important physiological processes in cell development and differentiation Different miRNA express differently at different developmental stages and different tissues Therefore the quantitative assaying of miRNA is important in both basic and applied research The All in One miRNA qRT PCR Reagent Kits use real time
6. PCR technology to quantitatively measure miRNAs The experimental procedure includes three major steps Figure 1 1 Adding poly A tails Poly A polymerase is used to add poly A tails to the 3 end of miRNAs 2 cDNA Synthesis At the same time M MLV RTase and a unique Oligo dT Adaptor primer reverse transcribes the poly A miRNAs The Universal Adaptor PCR primer in combination with a miRNA specific primer allows detection of specific miRNA 3 qPCR The All in One qPCR Mix containing SYBR Green specifically detects the reverse transcribed miRNA The miRNA specific forward primer is used with the Universal Adaptor primer When compared to traditional hybridization based miRNA detection methods such as Northern blot analysis the method provided by the All in One miRNA RT PCR Reagent Kits is faster more specific and sensitive and uses less sample material Key advantages e Provides efficient reverse transcription of miRNAs into cDNA in a single step e Delivers a precise quantitative and accurate measurement of miRNA expression profiles e __ Differentiates between mature and precursor miRNA e Co developed with validated primers miRNA clones and other tools used for functional studies of miRNA All in O ne miRNA qRT PCR Reagent Kits User Manual Total RNA Mature miRNA 3 Polyadenylation E mr Reverse transcription cDNA S o l Poly A polymerase Anneal oligo dT adaptor and RT reaction AAAAAAAAAA e T
7. VITTEETTTT T E an TTTTTTTTT cDNA template ready for qPCR All in One miRNA qPCR primer qPCR Assay 3 ee TTTTTTTTT Univesal Adaptor PCR Primer Figure 1 A graphic representation of the steps involved in the All in One miRNA qRT PCR reagent kits ll Related Products GeneCopoeia offers comprehensive solutions for studying human miRNAs A careful process of co development ensures that they work well together and provide robust and reproducible results Product Description All in One miRNA qPCR Primers Validated for robust reproducible and reliable quantitation of miRNA activity miProfile miRNA qPCR Arrays Genome wide or disease focused miRNA primer sets pre deposited in 96 or 384 well plates for qPCR profiling of miRNA expression miExpress Precursor miRNA Expression Clones Study miRNA regulation on target genes and proteins miTarget miRNA Target Validation Expression Clones Cross validate data using luciferase reporter genes OmicsLink Expression Ready ORF cDNA Clones All in One qPCR Mix Perform gain of function studies with expression ready clones Used in combination with miProfile miRNA qPCR Arrays and All in One miRNA First Strand cDNA Synthesis Kit to profile miRNA expression RNAzol RT RNA Isolation Reagent Easy isolation of mRNA microRNA or total RNA All in O ne miRNA qRT PCR R
8. e commercial products without prior written consent from GeneCopoeia 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 GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Manual If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia 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 GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia 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 GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose 10 All in O ne miRNA qRT PCR Reagent Kits User Manual GeneCopoeia is committed to providing our customer
9. eagent Kits User Manual lll Contents and Storage All in One miRNA First Strand cDNA Synthesis Kits Cat Nos AMRT 0020 and AMRT 0060 Contents Quantity Storage temperature conditions 2 5 Ulul 20 ul 20 C Stable for at least 12 months Alternatively the solution can also be stored at Poly A Polymerase 3 A0 80 C in aliquots Avoid repeated freezing thawing 20 ul 20 C Stable for at least 12 months RTase Mix 3x20 ul Alternatively the solution can also be stored at H 80 C in aliquots Avoid repeated freezing thawing 100 yl 20 C Stable for at least 12 months 5X PAP RT Buffer 5x00 Alternatively the solution can also be stored at H 80 C in aliquots Avoid repeated freezing thawing Spike in control 20 ul 20 C Stable for at least 12 months for use with miRNA qPCR 3x20 ul Alternatively the solution can also be stored at arrays only H 80 C in aliquots Avoid repeated freezing thawing dd H O imi 20 C Stable for at least 12 months RNase and DNase free imi Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing All in One miRNA qRT PCR Detection Kits Cat Nos AOMD Q020 and AOMD Q060 Primer Tm 64 5 GC content 50 Contents Quantity Storage temperature conditions 2 5 Ulul 20 ul 20 C Stable for at least 12 months Alternatively the solution can also be stored at Eoy A Polymera
10. escence detection temperature no more than the Tm value of the expected product If this does not work optimize and redesign the forward primer No signal Ct or Ct value is too high Check if there are PCR products to exclude the possibility of instrument detector malfunction Not enough PCR cycles For good sensitivity one should generally set up more than 35 PCR cycles but more than 45 cycles may result in too much background signals The amount of template may not be enough or the template may be degraded Use the highest concentration of diluted template samples possible to set up PCR At the same time avoid freezing and thawing samples repeatedly Amplification efficiency is low and PCR reaction conditions are not optimal Redesign primers and optimize reaction conditions VIII Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of all All in One miRNA qRT PCR Reagent Kits the Products If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia 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 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 manufactur
11. gent Kits User Manual e Ze R 0 997 E 103 9 f 4 z U 1 8 Zz pi AA f _ i 10 4 r T T T T T T T T i Log Starting Quandty copy number 0 3 lt SYBR E 103 9 RA20 997 slopes 3 233 yointe25 924 Figure 4 The amplification curve and standard curve generated from different amount of human brain total RNA as template and using All in One miRNA qRT PCR Detection Kit to detect hsa miR 124 expression level VII Trouble Shooting Guide miRNA sequence homology problems Because the sequence of miRNA is short and some have a high degree of homology the primer design can sometimes be tricky Thus one needs to fully consider the specificity problems when designing the miRNA forward primers Specifically for miRNAs that have a single nucleotide difference only the demand for specificity is higher for designing and synthesizing primers in addition to designing reaction conditions Confusion of amplification curves The fluorescence detection temperature may not be appropriate Adjust accordingly The set up position for samples may not be right Adjust accordingly Try to use 3 5 agarose gel electrophoresis to check the PCR products Check the purity of the primers using electrophoresis or use PAGE purified primers if the bands are diffused One may also use phenol chloroform extraction and ethanol precipitation methods to treat the primers before
12. ibed 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 and pipette tips if no RNase free and then autoclave to deactivate RNases RNase free consumables are available for purchase from many commercial sources Primer Design The reverse primer called Universal Adaptor PCR Primer Tm 64 5 GC 50 are provided in the All in One miRNA qPCR Mix Kit You may wish to design and make specific forward primers for your miRNA of interest or order from GeneCopoeia Please contact us for further information Since the length of miRNAs is generally between 18 24 nucleotides for some easy miRNAs a forward primer may be designed directly according to the sequence of the miRNA However for some potentially difficult miRNAs e g very high or very low Tm or highly homologous miRNAs or miRNAs from specific tissues e g tissues with high pre miRNA pri miRNA special primers may need to be designed to optimize the primer sequence in order to obtain specific amplification and avoid interference from pre miRNA pri miRNA IMPORTANT NOTES 1 Store kit at 20 C Avoid storage or leaving reagents at 4 C or room temperature 2 Mix reagents
13. ly If the total reaction volume is changed maintain each component in proper proportion Primer concentration should be in the range of 0 2 to 0 4 uM In general a PCR reaction using 0 2 uM primers produces good results The first strand cDNA should be diluted before using for the PCR reaction in order to avoid interference to the qPCR from the reverse transcription system ROX Reference Dye is used in Real Time PCR instruments that require ROX for calibration such as the ABI gPCR instrument Thoroughly mix the qPCR reaction solution add to PCR tubes and briefly centrifuge to make sure that all the reagents are in the bottom of the tubes The following standard 3 step method for the qPCR reaction is recommended example adapted from the iQ5 real time PCR detection system from Bio Rad Cycles Steps Temperature Time Deiection 1 Initial denaturation 95 C 10 min No Denaturation 95 C 10 sec No 40 Annealing Tm 2 C 20 sec No Extension 72 C At least 10 sec Yes Notes When using SYBR Green dye to monitor the qPCR reaction a melting curve analysis should be performed immediately after qPCR cycling For instructions consult the documentation for your qPCR instrument The following is an example adapted from the iQ5 real time detection system from Bio Rad Laboratories The conditions for your instrument may differ Temperature Range Heating Rate Constant Temperature Detection 65 C 95 C 0 5
14. s with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2013 GeneCopoeia Inc GeneCopoeia Inc 9620 Medical Center Drive Rockville MD 20850 Tel 301 762 0888 Fax 301 762 3888 Email inquiryr genecopoeia com Web www genecopoeia com GeneCopoeia Products are for Research Use Only Copyright 2013 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink All in One miProfile miTarget miExpress GeneCopoeia Inc SYBR Molecular Probes iQ 5 Bio Rad Trizol ABI ROX Life Technologies NanoDrop Thermo Scientific UMAOR062813 11
15. se 60 pl 80 C in aliquots Avoid repeated freezing thawing 20 ul 20 C Stable for at least 12 months RTase Mix 60 ul Alternatively the solution can also be stored at H 80 C in aliquots Avoid repeated freezing thawing 100 yl 20 C Stable for at least 12 months 5X PAP RT Buffer 300 ul Alternatively the solution can also be stored at H 80 C in aliquots Avoid repeated freezing thawing dd H O i ml 20 C Stable for at least 12 months Alternatively the solution can also be stored at RNase and DNase free ines 80 C in aliquots Avoid repeated freezing thawing 4 wal x2 20 C Stable for at least 12 months 2X All in One qPCR Mix ioe Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 80 ul 20 C Stable for at least 12 months 50X ROX Reference Dye 240 ul Alternatively the solution can also be stored at H 80 C in aliquots Avoid repeated freezing thawing 50 pM 20 ul i H 20 C Stable for at least 12 months Universal Adaptor PCR 60 ul H Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing All in One miRNA qRT PCR Reagent Kits User Manual IV Preparation 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 descr
16. thoroughly by gently inverting tubes several times avoiding bubbles and then briefly centrifuge before use 3 Following the procedure carefully to avoid contamination with RNases which can rapidly degrade RNA and lead to inconclusive results 4 Setup all reactions on ice to reduce risk of RNA degradation V Procedure Important note Follow the miProfile miRNA qPCR array user manual for the complete instruction when using the All in One miRNA first strand cDNA synthesis kit in combination with miProfile miRNA qPCR arrays and All in One qPCR mix 1 Reverse transcription of miRNA a Thaw template RNA on ice Thaw 5X PAP RT Buffer and ddH2O RNase DNase free at room temperature 15 25 C b Gently mix miRNA reverse transcription reagents by flicking to dissolve all reagents thoroughly Briefly centrifuge to collect residual liquid from the sides of the tubes and then place on ice c Prepare miRNA reverse transcriptase reaction solution Place RNase free reaction tubes on ice and then add the following reagents to a final volume of 25 ul All in O ne miRNA qRT PCR Reagent Kits User Manual Reagent Volume Quantity Total RNA 2 ug or small molecule RNA 100 ng 2 5 U ul Poly A Polymerase 1 ul RTase Mix 1 ul 5X PAP RT Buffer 5 ul 1X Spike in control Only comes in All in One miRNA first strand cDNA 1 pl synthesis kits dd H20 RNase DNase free To final 25 ul Total RNA must
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