Fast and straightforward expression analysis from small number of cells

May 20, 2015

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Company:

Life Technologies

Product Name:

Cell-to-Ct Power Sybr Green kit

Catalog Number:

A25600

I often perform mRNA expression analysis of a small (1-5) number of transcripts in different conditions of cell culture. The procedure of cell lysis, RNA extraction, reverse transcription and cDNA semiquantitative analysis by real time PCR is quite labor intensive and requires several sample transfers. With the Ambion Cell-to-Ct kit, the RNA extraction step is substituted by a lysis step that allows for straightforward reverse transcription in the same tube. This reduces the hands-on time, and allows for smaller samples.

Experimental Design and Results Summary

Application

Gene expression analysis

Starting Material

Colorectal cancer cell lines CaCO-2 and HT29

Protocol Overview

Cells are seeded at 10E5/ml and grown in 96-well plates for 48 hours in different culture conditions. After medium removal and wash with PBS, cells are lysed by directly pipetting 50 microliters of lysis buffer with DNaseI (optional) into wells, followed by incubation for 5 minutes at room temperature. Lysis is stopped by adding 5ul stop solution and mixing. After 2 minutes incubation, an aliquot (20% max vol) of the lysate is transferred to reverse transcription , and the remainder can be stored for future reference. Reverse transcription is assembled with a 2x buffer and a 20x enzyme mix and after a 65 minute thermal profile the cDNA is ready. Following the protocol you have enough cDNA for 12 qRT PCR data points (scalable). Altoghether it's less than a hour and a half from cell lysis to starting a qRT-PCR. This is where most of the value of the kit is. Samples are reduced in size, no cumbersome and labor intensive purification. The kit comes with the Power Sybr Green Master mix for the subsequent qPCR reaction.

Tips

Depending on the amount of cells you are using, the volumes can be scaled so as consume less product. I found this to yield consistent results with lysis volumes up to 40% of the initial requirement. If your amplicon is cross-exonic and DNaseI is not added, you might also amplify genomic DNA for concurrent analyses (Genotyping to name one),

Results Summary

qRT PCR analysis confirmed the results obtained by microarray profiling in a previous experiment

Additional Notes

For cells tightly attached to the substrate, like CaCO-2, I suggest performing the lysis by shaking the plate (I mount the plate on an Eppendorf thermomixer and set it to 600 rpm).

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Summary

The Good

Fast, eliminates a labor intensive step in routine expression analysis

The Bad

The reverse transcription requires adding water. Reagent concentration might be optimized so as to avoid this.

The Bottom Line

It's a very quick method that allows for more flexibility in your gene expression analysis

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