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Applied Biosystems™ TaqMan™ Advanced miRNA cDNA Synthesis Kit

Catalog No. A28007
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A28007 50 cDNA synthesis reactions
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Description

Kit uses 3' poly-A tailing and 5' ligation of an adaptor sequence to extend the mature miRNAs present in the sample on each end prior to reverse transcription.

The Applied Biosystems TaqMan Advanced miRNA cDNA Synthesis Kit uses a universal reverse transcription (RT) chemistry to prepare the cDNA template for use with TaqMan Advanced miRNA Assays for detection and quantification of mature miRNAs in biological samples. The kit is designed to perform 50 cDNA synthesis reactions, with theresulting cDNA sufficient for up to 600 qPCR reactions, allowing extensive downstream analysis.

Features of the TaqMan Advanced miRNA workflow include:

  • Sensitivity: high sensitivity allows for quantification of low-expressing miRNA targets, especially in biological fluids such as plasma, serum, and tissue
  • Flexibility: the universal RT chemistry generates universal cDNA templates for all miRNAs present in the sample, allowing for flexibility in study design
  • Sample savings: requires only 2 μL of plasma/serum sample input and generates enough cDNA for 600 qPCR reactions (20 μL) or 1200 qPCR reactions (10 μL)
  • Specificity: 5' ligation step allows for single base pair discrimination along entire miRNA sequence, including difficult-to-detect mismatches on 5' end
  • Gold-standard TaqMan chemistry: This kit is designed for use with TaqMan Advanced miRNA Assays for qPCR, which draw on Thermo Fisher Scientific's industry-leading bioinformatics assay design pipeline and validation system to help ensure high specificity and minimal cross-reactivity, even for closely-related miRNA families.

The TaqMan Advanced miRNA cDNA Synthesis Kit uses 3' poly-A tailing and 5' ligation of an adaptor sequence to extend the mature miRNAs present in the sample on each end prior to reverse transcription. Universal RT primers recognize the universal sequences present on both the 5' and 3' extended ends of the mature miRNAs. All mature miRNAs in the sample are reverse transcribed to cDNA.

In order to improve detection of low-expressing miRNA targets, the cDNA is then amplified using the Universal miR-Amp Primers and miR-Amp Master Mix to uniformly increase the amount of cDNA for each target, maintaining the relative differential expression levels. Unlike traditional pre-amplification, the Universal miR-Amp Primers recognize the universal sequences added to all mature miRNAs on the 5' and 3' ends, ensuring that there is no amplification bias.

Note: the TaqMan Advanced miRNA cDNA Synthesis Kit is not compatible with first generation TaqMan MicroRNA Assays for qPCR. For reverse transcription of miRNA targets using miRNA-specific RT primers, please see the TaqMan MicroRNA Reverse Transcription Kit.

Order Info

Note: the TaqMan Advanced miRNA cDNA Synthesis Kit is not compatible with first generation TaqMan MicroRNA Assays for qPCR. For reverse transcription of miRNA targets using miRNA-specific RT primers. Shipping Conditions: Dry Ice

Specifications

Content And Storage • 10X PolyA Buffer
• ATP, 10mM
• Poly A Enzyme, 5 U/μL
• 5X Ligase Buffer
• RNA Ligase, 10 U/μL
• 50% PEG 8000
• 25X Ligation Adaptor
• 10X RT Enzyme Mix
• 20X Universal RT Primer
• dNTP Mix, 100mM
• 20X miR-Amp Primer Mix
• 5X RT Buffer
• 2X miR-Amp Master Mix

Store all at -5 to -30°C, except 2X miR-Amp Master Mix which is stored at 2–8°C.
Detection Method Primer-probe
GC-Rich PCR Performance High
PCR Method qPCR
Reaction Speed Standard
For Use With (Equipment) 7500 System, 7500 Fast System, 7900HT System, StepOne™, StepOnePlus™, ViiA™ 7 System, QuantStudio™ 3, QuantStudio™ 5, QuantStudio™ 6 Flex, QuantStudio™ 7 Flex, QuantStudio 6 Pro, QuantStudio 7 Pro, QuantStudio™ 12k Flex, QuantStudio™ Absolute Q Digital PCR System
Optimal Reaction Temperature 42°C
Product Line TaqMan
Product Type cDNA Synthesis Kit
Quantity 50 cDNA synthesis reactions
Reverse Transcriptase SuperScript™ III
Shipping Condition Dry Ice
For Use With (Application) miRNA analysis
No. of Reactions 50 cDNA synthesis reactions / 600 qPCR reactions
Reaction Format Separate Components
Sample Type RNA
Volume 10 μL
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Frequently Asked Questions (FAQs)

Can I purchase any of the components within the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) separately?

No, none of the single components within the kit are available as stand-alone. They only can be ordered within the complete kit.

Do you have a modified protocol for the TaqMan MicroRNA Cells-to-CT Kit (Cat. No. 4391848) to make it compatible with the TaqMan Advanced miRNA Assays?

The workflow to use the Cell-to Ct kit with the TaqMan Advanced miRNA Assays is to use 2 µl of the lysate post cell lysis as input into the TaqMan Advanced miRNA cDNA synthesis Kit.
What is the concentration of the probe and primer in the 20X TaqMan Advanced miRNA Assay (Cat. No. A25576)?

Unfortunately, these concentrations cannot be disclosed. Use the assay at a final concentration of 1x in the total reaction volume.
Do you have any suggestion on how to use the TaqMan Advanced miRNA Assay with Fluidigm system?

Our recommendation for this process would be to carry out a cDNA synthesis according to the TaqMan Advanced miRNA protocol, followed by scaling down QPCR reaction in accordance with the Fluidigm protocol.
At what concentration should I add the spike-in control into my sample for the TaqMan Advanced miRNA Assay?

The final concentration of the spike-in control in your test sample should be 1–10 pM.
At what point can I add the spike-in miRNA in the TaqMan Advanced miRNA Assay workflow?

The spike-in miRNA should be added to the sample after addition of denaturant and prior to addition of chloroform and phase separation.
Does the reverse transcription primer in the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) contain degenerate base at the 3’ end?

Yes it does. This is the schematic map of the RT primer: 3’-Anchor base - Poly T tract - Adaptor sequence-5’.
Can I detect miRNA and mRNA simultaneously with the TaqMan Advanced chemistry?

Yes, this is possible. We have a protocol for the simultaneous detection of miRNA and mRNA on TaqMan Array cards. Please see the following Application Note for more information.
The TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) utilizes a universal RT primer which would reverse transcribe not only miRNA but also mRNA (as the primer is polyA tail based). How do you prevent reverse transcribing mRNA?

TaqMan advanced miRNA cDNA synthesis kit will also allow mRNA to be reverse transcribed (because of PolyA tail). However, the miR-Amp step is very specific to miRNA and does not help in amplifying mRNAs.
I see some precipitation in the 50% PEG 8000 tube of the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007). I defrosted it, however, the pellet remains. Can I use this solution?

Precipitation in 50% PEG is very common, especially at low temperatures. You can warm up the PEG tube at 65°C to bring it back into solution and cool it on ice before using it.
How can I improve the sensitivity of the reaction with TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) to enable detection of low copy number miRNA?

We recommend increasing the number of miR-Amp cycles from 14 to 18. This might help detect targets which are present in very small copy numbers in the sample.
For improving the sensitivity of the reaction can I reduce the total reaction volume by decreasing the volume of RNase-free water in the miR-Amp step of the TaqMan Advanced cDNA Synthesis Kit (Cat. No. A28007)?

Decreasing the amount of water in the miR-Amp reaction is not recommended since this would change concentration of master mix.
Do I need to dilute the cDNA prepared with the TaqMan Advanced cDNA Synthesis Kit (Cat. No. A28007) for the qPCR reaction?

Dilution of the miR-Amp product is essential to prevent carry over of mir-Amp primers into the PCR reaction. These can produce non-specific amplification and/or a high background noise.
Have you ever run the cDNA prepared with the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) on a gel for the +RT and –RT reactions? What would these look like?

The bands seen in –RT are not of cDNA but of the adapter ligated and PolyA tailed miRNA. Size of both the hybrid molecules i.e., the miRNA and cDNA would be the same and hence they would both would be detected on the gel. There is no way to discriminate between the two on the gel. See figure on page 23 of the User Guide for an illustration of how the polyA tailing and adapter ligation as well as the reverse transcription to generate a miRNA-cDNA hybrid work.
Have you tested the PureLink RNA Mini Kit (Cat. No. 12183018A) or PureLink miRNA Isolation Kit (Cat. No. K157001) for extraction of RNA with the TaqMan Advanced miRNA Assays?

These kits have not been tested. However, these kits should work with the TaqMan Advanced miRNA Assays. We recommend checking the quality and quantity of small RNAs post-isolation by NanoDrop and Bioanalyzer.
What is the length of the miR-Amp product from the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007)?

The size of the miR-Amp product is 118 bp on average depending on the length of the miRNA.
What is the length of the 5’ adapter which gets ligated to the mature miRNA in the for TaqMan Advanced miRNA cDNA synthesis reaction?

The 5' adaptor is 41 bp.
Can I use more than 10 ng of total RNA in making cDNA for TaqMan Advanced miRNA Assay and the array card?

We do not recommend going above 10 ng of total RNA. Above 10 ng, the reagents in the cDNA synthesis kit become limiting in the reaction. The performance will decrease if higher input amounts greater than 10 ng are added.
Is the cDNA made from the TaqMan Advanced miRNA cDNA Kit (Cat. No. A28007) compatible with SYBR Green?

The cDNA made from A28007 is not compatible with SYBR chemistry. We do not sell the primers needed for qPCR with SYBR green chemistry separately.
The Guidelines for RNA Input section of the user guide of the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) recommends using 2 µL of sample eluent. Is this volume the total RNA or liquid samples (blood, serum, plasma)?

This kit works with purified RNA. Therefore 2 µL is the volume of total RNA purified from the above liquid samples. If RNA can be quantified, use 1-10 ng total RNA per reaction.
I stored the miR-Amp Master Mix from the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) at -20°C. Can I still use it?

It should be OK if the miR-Amp Master Mix was stored one time at -20°C. The kit is shipped on dry ice. Please store it at 4°C moving forward and do not return it to -20°C.
At what temperature should I store the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007)?

The above kit has multiple components; most of them are stored at -20°C. The 2x miR-Amp Master Mix is stored at 2-8°C. For details please see page 7 of the user guide.
The color of the 2x miR-Amp Master Mix in the TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007) is blue. Is this correct?

The color of miR-Amp Master Mix is blue as it has a dye to track the reaction.
Is TaqMan Advanced miRNA system compatible with plant miRNA?

The plant miRNAs with 2′-O-methylation at its 3’ end should still work with the TaqMan Advanced miRNA system, albeit with reduced sensitivity. The miRNA should have a phosphate at its 5’ end for the TaqMan Advanced chemistry to work.
How does TaqMan Advanced miRNA (Cat. No. A25576) work?

The TaqMan Advanced miRNA workflow comprises five steps. Steps 1-4 are completed using the TaqMan Advanced miRNA cDNA Synthesis kit (Cat. No. A28007). Please check the protocol in the TaqMan Advanced miRNA Assays User Guide. Step 5 is completed using the TaqMan Advanced miRNA Assays (Cat. No. A25576) together with the TaqMan Fast Advanced Master Mix (Cat. No. 4444556, sold separately). Briefly, the steps are as follows:

  • After isolation of total RNA, using a method that preserves small RNAs, Poly A polymerase is used to add a polyadenosine (polyA) tail on the 3’ end of all of the miRNAs in the sample.
  • In the next step, an oligonucleotide adaptor is ligated to the 5’ end of each of the miRNAs. This reaction is dependent on the presence of a 5’ phosphate, which is only present on mature miRNAs. Any exogenous controls or spike-ins must also have this 5’ phosphate in order to be accurately quantitated.
  • A Universal RT primer binds to the polyA tail of the miRNA to reverse transcribe the RNA into cDNA. This universal RT step will convert all of the mature miRNAs in the sample to cDNA.
  • Universal amplification (miR-Amp) reaction takes place, wherein, the universal forward and reverse primers amplify the cDNA for all miRNA targets in the sample. The miR-Amp step improves quantification of low expressing miRNAs and miRNAs collected from dilute samples such as body fluids.
  • miRNA-specific primers and TaqMan probe are used to quantitate each miRNA of interest using individual qPCR assays called TaqMan Advanced miRNA Assay. These assays work specifically with TaqMan Advanced miRNA and cannot be used with the original TaqMan Micro RNA Assay chemistry.
What reagents and/or kits are required for the TaqMan® Advanced miRNA Assay workflow using the TaqMan® Advanced miRNA Human A and B cards (Cat. No. A31805)?

All materials required for TaqMan® Advanced miRNA Assay workflow using TaqMan® Advanced miRNA Human and B cards (Cat. A31805) can be found on pages 9-11, in the TaqMan® Advanced miRNA Assays User Guide.

Reagents and kits required for the TaqMan® Advanced miRNA Assay workflow include, but are not limited to:

• RNA Isolation Kit (see recommended kits on page 9)
• TaqMan® Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007)
• TaqMan® Fast Advanced Master Mix (Cat. No. 4444557)

Please refer to the user guide linked above for a full list of other materials and equipment required for the workflow.

Do you offer an alternative for TaqMan Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007)?

If you are performing your analysis with the TaqMan Advanced miRNA Assay (Cat. No. A25576), we do not offer an alternative and the cDNA has to be synthesized with that kit.

For Research Use Only. Not for use in diagnostic procedures.