miRNAssay™ qPCR RT Master Mix SuperPrep™ miRNAssay™ Cell Lysis & RT Kit for qPCR│TOYOBO PRODUCTS LIST│Research Reagents │TOYOBO

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  • miRNAssayTM qPCR RT Master Mix/
    SuperPrepTM miRNAssayTM Cell Lysis & RT Kit for qPCR

DESCRIPTION

  • miRNAssay™ qPCR RT Master Mix [Code No. MIR-101] is a real-time PCR reverse transcription reagent for miRNA developed using the high-efficiency reverse transcriptase ReverTra Ace™. It is optimized for reverse transcription reactions using stem-loop RT primers and can synthesize cDNA from miRNA with high specificity and sensitivity.
    SuperPrep™ miRNAssay™ Cell Lysis & RT Kit for qPCR [Code No. MIR-201] consists of the above miRNA-specific reverse transcription master mix and lysis reagents for cultured cells and exosomes. This kit enables high-quality cDNA synthesis from cultured cells or recovered exosomes without purification of RNA.

    ※Stem-loop RT primer, Real-Time PCR Reagent, Forward (Fwd) Primer, Reverse (Rev) Primer, and Probe are not included in this product.
    For real-time PCR, we recommend using our highly efficient real-time PCR reagents THUNDERBIRD™ Next SYBR™ qPCR Mix [Code No. QPX-201] or THUNDERBIRD™ Next Probe qPCR Mix [Code No. QPX-101].

Features

  • - Premix cDNA synthesis reagent
    Reverse transcription reagents are premixed. This kit simplifies reagent preparation and provides stable results by reducing variations.
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  • - High specificity
    cDNA is specifically synthesized from mature miRNAs with an optimized composition of stem-loop RT primers.
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  • - Broad dynamic range
    cDNA can be synthesized with high efficiency and specificity from low-copy targets to high-copy targets.
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  • - Excellent reagent compatibility
    This kit can be used in combination with various real-time PCR reagents.
    Commercially available stem-loop RT primers are also applicable.
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  • - cDNA synthesis without RNA purification from cultured cells / exosomes
    SuperPrep™ miRNAssay™ contains lysis solution reagent for cultured cells and exosomes. miRNAs can be converted to cDNA without RNA purification.

Details

Storage condition

Store at -20℃

Components

miRNAssay™ qPCR RT Master Mix MIR-101 MIR-101T
Size 200 Rxns*1 40 Rxns*1
5×RT Master Mix 400 μL 80 μL
5×RT Master Mix no-RT Control 40 μL 10 μL
Nuclease-free Water 1.1 mL x 2 440 μL
SuperPrep™ miRNAssay™ Cell Lysis & RT Kit for qPCR MIR-201 MIR-201T
Size 100 Rxns*2 20 Rxns*2
5×RT Master Mix 800 μL 160 μL
5×RT Master Mix no-RT Control 80 μL 16 μL
Nuclease-free Water 1.7 mL x 2 440 μL
Lysis Solution 6.5 mL 1.3 mL
gDNA Remover 33 μL 10 μL
RNase Inhibitor 110 μL 22 μL

*110μL RT reaction　　*240μL RT reaction

*Stem-loop RT primer and primer-probe set are not supplied with this kit.

DETECTION PRINCIPLE

This product is a reagent that reverse-transcribes miRNA using a looped RT primer (stem-loop RT primer). Stem-loop RT primers allow miRNA to be efficiently reverse-transcribed and their length can be extended simultaneously with reverse transcription.

Application Data

APPLICATION DATA

Example 1.Confirmation of specificity using highly homologous miRNAs

Yeast Total RNA 100 ng was mixed with 105 copies of let-7a, let-7g, and miR-98 synthetic RNA, and cDNA synthesis was performed using miRNAssay™ qPCR RT Master Mix and another company's reagent. Real-time PCR was performed using the synthesized cDNA as a template, and the relative detection efficiency was compared.
Relative detection efficiency was calculated from Ct differences between perfectly matched and mismatched targets, assuming 100% efficiency for the perfect match. This product has been shown to identify even highly homologous miRNAs with high accuracy.

Example 2.cDNA synthesis from 10-fold dilution series (1pg~100ng) of total RNA

Using miRNAssay™ qPCR RT Master Mix and let-7a-specific stem-loop RT primer, cDNA was synthesized from a 10-fold dilution series (1 pg to 100 ng) of total RNA derived from HeLa cells. 1/10 of the reverse transcription solution was subjected to real-time PCR to confirm quantitative performance. Similarly, cDNA of let-7a was synthesized using another company's reverse transcription reagents, and real-time PCR analysis was performed.
In this product, no amplified signal was detected in no-RT Control, which is RT(-) control, and quantitative detection of let-7a was possible in a wide range from 1 pg to 100 ng.

Example 3.Detection of miRNAs from various cell lysates

Using the SuperPrep™ miRNAssay™ Cell Lysis & RT Kit for qPCR, cell lysates were prepared from 5×104 cells of K562 (human chronic myeloid leukemia cell line), HepG2 (human hepatoma-derived cell line), Jurkat (human Leukemia T cell-derived cell line), HeLa (human cervical carcinoma-derived cell line), and A431 (human epithelioid cell carcinoma-derived cell line). cDNA synthesis of 6 miRNAs (let-7a, d, e, f, g, miR-98) was performed. In addition, cDNA was similarly synthesized from RNA purified from cultured cells. Real-time PCR analysis was performed using each cDNA as a template and THUNDERBIRD™ Next SYBR™ qPCR Mix [Code No. QPX-201].
High correlations were observed between cDNA synthesized from lysate and cDNA synthesized from purified RNA for 6 types of targets and 5 types of cell types. This product enables miRNA expression analysis by real-time PCR without complicated RNA purification.

Example 4.Detection of miRNAs from exosome lysate

Using SuperPrep™ miRNAssay™ Cell Lysis & RT Kit for qPCR, exosomes collected from the culture supernatant of HeLa cells (culture supernatant 2 mL) were lysated and cDNA was synthesized with specific stem-loop RT primers for each of 9 different miRNAs (let-7a, c, d, e, f, g, i, miR-21, 103). cDNA was similarly synthesized from RNA purified from exosomes. Real-time PCR analysis of each cDNA confirmed a good correlation between exosome lysate and purified RNA.
By using this kit, the time and loss of RNA purification can be eliminated and accurate analysis can be performed.