29th November 2024

qScript™ microRNA cDNA Synthesis Kit

 

The qScript microRNA cDNA Synthesis Kit is an optimized reagent system that reverse transcribes small single-stranded RNA into 5′-labeled cDNA using either total RNA or miRNA enriched samples.

Single-stranded RNA is first polyadenylated by poly(A) polymerase before reverse transcription into universal cDNA using high performance qScript RT with a proprietary adapter oligo(dT) primer.  The universal cDNA template enables simple and cost-effective microRNA profiling when used together with wet-lab validated PerfeCTa microRNA Assays, PerfeCTa Universal PCR Primer and PerfeCTa SYBR Green SuperMix.

This complete kit includes positive (human) control primer, SNORD44, that can be used to quantitate ubiquitously expressed small nucleolar RNA. In addition, the kit contains 20% extra poly(A) polymerase reaction buffer and microRNA cDNA Reaction Mix to perform (-) poly(A) polymerase and (-) reverse transciptase control reactions.

  • Sensitive reverse transcription of small, single-stranded RNA
  • Labeled universal cDNA supports seamless qPCR analysis with PerfeCTa miR Assays or user-defined assay primer (e.g. piRNA, lncRNA) together with PerfeCTa Universal PCR Primer
  • Optimized for use with PerfeCTa SYBR Green SuperMix
  • For more information on Quantabio’s miRNA System click here.

Contents

  • Poly(A) Tailing Buffer (5X)
  • Poly(A) Polymerase
  • MicroRNA cDNA Reaction Mix
  • qScript Reverse Transcriptase
  • PerfeCTa Universal PCR Primer
  • PerfeCTa Human Positive Control Primer
  • Nuclease-Free Water

Performance Data

Quanta microRNA Technology

microrna_detection

The qScript microRNA cDNA Synthesis Kit is suitable for use with total RNA or preparations pre-enriched for microRNAs.  A poly(A) tail is added to microRNAs followed by cDNA synthesis using an adapter primer and qScript RT in a single-tube reaction. The resulting cDNA is ready for qPCR amplification with a Universal PCR Primer and your choice of microRNA Assays. This unique single-tube system provides ease-of-use and efficient cDNA synthesis for quantification of microRNAs with a high degree of sensitivity and specificity. PerfeCTa™ SYBR Green SuperMixes ensure compatibility with any real-time thermal cycler.

 

microRNA Detection Range

mir-1_curves

Detection of rare microRNAs
Quanta’s microRNA profiling system provides linear detection and quantification of microRNAs across total-RNA input levels spanning six orders of magnitude. This means microRNAs will be detected when present even when tissue is scarce or the microRNAs are rare.

 

Quanta – let-7a

microquanta_vs_qiagen_let-7a

qScript; microRNA Quantification System Comparison to Competitor (hsa-let-7a)
microRNA cDNA synthesis was performed starting with log-fold dilutions of human brain total RNA (from 1.0 µg to 10 pg) using the qScript microRNA cDNA Synthesis Kit (Quanta) or the miScript Reverse Transcription Kit (Qiagen). 1/100th of each microRNA cDNA reaction was used in a 20 &microL qPCR amplification reaction specific for hsa-let-7a using each manufacturers validated microRNA assay, qPCR mix and recommended protocol. Averaged plots for triplicate qPCR reactions for each input quantity are shown. Cycling conditions for the miScript Primer Assay with the miScript SYBR Green PCR Kit (Qiagen) consisted of 94°C, 15 min followed by 40 cycles of 94°C, 15s; 55°C, 30s and 70°C, 30s. Cycling conditions for the PerfeCTa microRNA Assay and PerfeCTa SYBR Green SuperMix; (Quanta) consisted of 95°C, 2 min followed by 40 cycles of 95°C, 5s and 60°C, 30s.

 

Quanta vs ABI

mir quanta_vs_abi_mir-124_1

qScript microRNA Quantification System Comparison to ABI (hsa-miR-124)
MicroRNA cDNA synthesis was performed starting with log-fold dilutions of human brain total RNA (from 1.0 µg to 100 pg) using the qScript MicroRNA cDNA Synthesis Kit (Quanta) or the TaqMan microRNA Reverse Transcription Kit (ABI). 1/100th of each microRNA cDNA reaction was used in a 20 µL qPCR amplification reaction specific for hsa-miR-124 using each manufacturers validated microRNA assay, qPCR mix and recommended protocol. Averaged plots for triplicate qPCR reactions for each input quantity are shown. Cycling conditions for the TaqMan MicroRNA Assay with TaqMan Universal PCR Master Mix (ABI) consisted of 95°C, 5 min followed by 40 cycles of 95°C, 15s; 60°C, 1 min. Cycling conditions for the PerfeCTa MicroRNA Assay with PerfeCTa SYBR Green SuperMix (Quanta) consisted of 95°C, 2 min followed by 40 cycles of 95°C, 5s and 60°C, 30s.

Storage conditions

The kit components are stable for up to one year when stored at or below -20°C.

Resources

Ordering

Cat# 95107-025 25 x 20 µl rxns
Cat# 95107-100 100 x 20 µl rxns