Super Fast Realtime Expression Analysis from Purified RNA or Cells

January 26, 2016

Overall

Quality of Results

Ease-of-Optimization

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Company:

Ambion

Product Name:

Single Cell-to-CT™ Kit

Catalog Number:

AM1729

This is a versatile kit for immediate gene expression analysis, results are obtained within the hour.

Experimental Design and Results Summary

Application

Gene expression, genomic analysis, DNA removal

Starting Material

Cultured cells

Protocol Overview

Cells are harvested and washed with PBS, then lysis buffer is applied, DNAse removal is performed (optional) and stopped after 5' by a proteic inhibitor. The lysate is then added to a mix of 5x reaction buffer and TaqMan assay of interest. Reverse transcription (5') and realtime PCR (fast cycle ~30') are performed in the same tube yielding results in less than a hour.

Tips

The lysate can be used for DNA analysis, prior to DNAse treatment. DNase and Stop Solution in the kit can also be used to remove DNA contamination on RNA preps, whereby additional purification after DNase treatment would lead to substantial loss of material.

Results Summary

My RNA samples were scarce and heavily contaminated with DNA. Quantifying with nanodrop was inaccurate. I used RNA from an abundant source to calibrate sensitivity of the kit. Dilutions of up to 2pg yielded a curve on real-time PCR for the housekeeping gene GUSB (fig. a), and I was able to quantitate the RNA content of my samples (blue squares, fig.b)

Additional Notes

The DNAse treatment is higly efficient and is stopped by the addition of the stop solution. I premixed the stop solution to DNase and checked it for activity on plasmid DNA. The DNA remained intact.

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Summary

The Good

The kit allows for rapid analysis of DNA and RNA from small amount of cells in less than one hour and with limited steps

The Bad

Price per sample is not low

The Bottom Line

When scaled and optimized, this kit can be preferred over reporter assays for transcript quantification in gene regulation experiments

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