Code No. MIR-101T, MIR-101/Code No. MIR-201T, MIR-201
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].
Store at -20℃
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.
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.
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.
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.
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.
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.