An optimised direct lysis method for gene expression studies on low cell numbers.

Anh Viet-Phuong Le,Erik W Thompson,Tony Blick,Dexing Huang,Alexander Dobrovic

doi:10.1038/srep12859

Abstract

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

Highlights

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells
We demonstrated that IGEPAL CA-630/bovine serum albumin (BSA) lysis solution performed the best and was superior to a commercially available column-based methodology for small numbers of cells
We tested the ability of five different lysis solutions to lyse a small number of MDA-MB-468 breast cancer cells, and their compatibility with downstream reverse transcription (RT)-qPCR

Summary

Introduction

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. Direct lysis methods offer a simpler, cheaper and faster approach of preparing RNA to study gene expression profiles of CTCs, especially for single cells or small numbers of cells. A recent study by Svec et al compared 17 different lysis solutions (including a IGEPAL CA-630 based lysis buffer) for a small number of astrocytes and found that a lysis solution containing bovine serum albumin (BSA) in water gave the best RT-qPCR performance[16]

Methods

Results

Conclusion