Rapidly tag and convert all small RNAs into detectable cDNA for qPCR
Simple and robust procedure
Powerful technology to convert enough RNA to cDNA for up to 5,000 qPCR measurements
Validate newly discovered MicroRNAs
Detect and Quantitate siRNAs in knock-down experiments
Amenable to high-throughput screening of clinical samples including laser-captured micro dissected specimens
Three reference assays included in each kit: Human U6, Mouse U6 and Human/Mouse miR-16
Simple and effective to use
Efficient, Sensitive and Accurate
Highly efficient poly(A) tailing and reverse transcription in a single reaction tube provides uniform cDNA synthesis of microRNAs. The optimized reaction conditions and buffer components maximize cDNA yield using anywhere from several micrograms to picograms of input total RNA. The universal 3’ tag sequence incorporated during reverse transcription enables the ease of scalable and accurate microRNA expression analysis by qPCR. Profile 1000 different microRNAs from a single reverse transcription reaction.
Sensitive
Start with total, Trizol-extracted RNA or fractionated small RNA samples. Achieve measurements from several micrograms to picograms of input RNA with excellent accuracy. Detect microRNA expression changes in a dynamic range of at least 6 log-fold differences.
Accurate
First strand cDNAs were synthesized using 500 ng total RNA from 18 different human tissues: adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, and thymus. The QuantiMir cDNAs from the 18 human tissues were balanced to yield equal Ct values for the U6 snRNA normalizing transcript (Below in Green bars). Real-time PCR results demonstrated this snRNA is uniformly expressed across the 18 tissues examined. Assays specific for microRNA miR-1 demonstrate specific Heart and Skeletal expression (Below in Red bars) and assays for microRNA miR-122 clearly show specific Liver expression (Below in Blue bars).
Numerous miRNAs has been found to have links with some types of cancer. Expression profiling of microRNAs can assist in identifying the type and severity of the oncogenic state and microRNA expression "signatures" can be used to develop diagnostics and therapeutics.
Measure both siRNA and mRNA knockdown in a single QuantiMir cDNA sample
Quantitate siRNAs and mRNA knockdown using QuantiMir: The use of sh/siRNAs to lower the levels of endogenous messenger RNA expression is a powerful and effective tool to study the roles of a targeted transcript in biological systems. Real-time qPCR has become the gold standard in accurately measuring the knockdown effects of specific sh/siRNAs in these experiments. The means of assessing the expression levels of sh/siRNA input has been typically achieved through laborious and nonquantitative Northern blot analyses. Here we present a new method to quantitate both the expression levels of sh/siRNA and the knockdown effect on the targeted messenger RNA transcript using a passive lysis buffer coupled with the QuantiMir kit cDNA synthesis and Real-time qPCR.
After transfection of siRNA or shRNA expression constructs, the cells are removed from the transfection plate using standard trypsinization. The cells are pelleted and subsequently lysed using 100 ul of SBI’s Cells-to-Cts passive lysis Buffer system. A 5 ul aliquot of the lysed cell suspension is then input directly into the QuantiMir RT reaction. The unique technologies in the QuantiMir RT kit enables the conversion of small RNA into detectable cDNA while simultaneously converting messenger RNA into cDNA. Accurate measurement of sh/siRNAs and the messenger RNA transcript in knockdown experiments can be accomplished using the cDNA created from this single RT reaction. The QuantiMir RT reaction generates enough cDNA suitable for numerous qPCR reactions. Shown below is an example of a timecourse study using anti-p53 shRNA-directed knockdown of the endogenous p53 mRNA transcript. From the same QuantiMir cDNA, both the p53 siRNA (Orange bars) and the p53 mRNA transcript (Blue line) were measured by qPCR.
