miRNA cDNA第一链合成试剂盒 (含 gDNA 去除试剂，茎环法)

miRNA cDNA First Strand Synthesis Kit (contains gDNA removal reagent, stem-loop method)

Product Code: AG11745

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¥700

Product Description

This is a reverse transcription kit for synthesizing miRNA first strand cDNA by stem-loop method. The 5X gDNA Clean Reaction Mix II effectively removes residual genomic DNA (gDNA) from RNA at 42°C for 2 min to improve the accuracy of results. This product is suitable for reverse transcription of miRNA-containing samples such as Total RNA or Small RNA. The cDNA synthesized with this product is suitable for chimeric qPCR analysis and is recommended to be used with SYBR Green. Pro Taq HS Premixed qPCR Kit II (Code. AG11702); for high demand for elimination of primer dimers, SYBR Green can be used. Pro Taq HS Premixed qPCR Kit IV (Code. AG11746) for superior results.

Product Advantages

① 5X gDNA Clean Reaction Mix II contains a gDNA removal reagent that rapidly removes residual gDNA from RNA templates.
② This product can reverse transcribe samples containing miRNA such as Total RNA or Small RNA.
③ The cDNA obtained by inversion of this product can realize accurate quantification of mature miRNA with high sensitivity and specificity.

Product Composition
individual parts making up a compound norm
5X gDNA Clean Reaction Mix II 100 μl
5X miRNA RT Buffer (Stem-loop)*1 200 μl
miRNA RT enzyme mix (Stem-loop) *2 100 μl
RNase free water 1 ml

*1: Contains dNTP.
*2: contains Evo M-MLV RTase and RNase Inhibitor.

Preservation and transportation
Storage temperature: -20℃ storage
Transportation temperature:Dry ice transportation or -20°C ice pack transportation.
Example 1

200 ng of mouse gDNA was added to mouse liver Total RNA (200 ng, 20 ng, 2 ng, 200 pg, 0 pg, respectively), and gDNA was removed using this product (red curve in the figure below). At the same time, 5X gDNA Clean Reaction Mix II was inactivated with the product as a gDNA control (green curve in the figure below). Then, reverse transcription was performed and 2 μl of the cDNA stock solution was taken from the cDNA obtained from the reverse transcription as a template, and the cDNA was analyzed by SYBR Green. Pro Taq HS Premixed qPCR kit (AG11701) for qPCR amplification to detect mouse GAPDH. Quantitative instruments used: ABI QuantStudioTM 5 Real-Time PCR Systems. the results are shown below:

The results are shown above:
1、Working curve R2= 1.0000 and the amplification efficiency was 106.21 TP3T.
2、This product can effectively remove the residual gDNA in RNA, and the melting curve obtained has a single peak shape with good amplification specificity.
3、The product has high reversal efficiency and can perform accurate quantification in a wide template range. The cDNA amplification curves obtained from 200 ng ~ 200 pg RNA reverse transcription show a good linear relationship.

Example 2

Mouse liver Total RNA (1 μg, 100 ng, 10 ng, 1 ng, 100 pg, 0 pg, respectively) was used as a template for reverse transcription experiments using this product, and 2 μl of cDNA stock solution was taken from the cDNA obtained by reverse transcription as a template, which was then analyzed by SYBR Green. Pro Taq HS Premixed qPCR Kit II (AG11702) for qPCR amplification to detect mouse rno-miR-802-5p。
Quantitative instrument used: ABI QuantStudioTM 5 Real-Time PCR Systems. the results are shown below:

The results are shown above:
1、Working curve R2= 0.9945 and the amplification efficiency is 100.41 TP3T.
2. The amplification curves in the range of 1 μg ~ 100 pg RNA concentration showed good linearity.
3、Single melting curve peak type, good amplification specificity.

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