TaqMan™ Advanced miRNA cDNA Synthesis Kit - FAQs

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

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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.

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Unfortunately, these concentrations cannot be disclosed. Use the assay at a final concentration of 1x in the total reaction volume.

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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.

The final concentration of the spike-in control in your test sample should be 1–10 pM.

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The spike-in miRNA should be added to the sample after addition of denaturant and prior to addition of chloroform and phase separation.

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Yes it does. This is the schematic map of the RT primer: 3’-Anchor base - Poly T tract - Adaptor sequence-5’.

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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.

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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.

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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.

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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.

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Decreasing the amount of water in the miR-Amp reaction is not recommended since this would change concentration of master mix.

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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.

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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.

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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.

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The size of the miR-Amp product is 118 bp on average depending on the length of the miRNA.

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The 5' adaptor is 41 bp.

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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.

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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.

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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.

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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.

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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.

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The color of miR-Amp Master Mix is blue as it has a dye to track the reaction.

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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.

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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.

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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.

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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.