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

Le AV, Huang D, Blick T, Thompson EW, Dobrovic A - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia [2] Translational Genomics &Epigenomics, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of five different lysis solutions for small number of MDA-MB-468 breast cancer cells.Three replicate samples of approximately one hundred cells were subjected to each different lysis solution, RT-qPCR was run and threshold cycle (Ct) values were determined for RPL32 and EGFR. The earliest Ct values of IGEPAL CA-630/BSA indicate that it performed the best. Conversely, the latest Ct values for samples lysed in water indicate that it performed the worse. Lysis with BSA alone led to earlier Ct values than those obtained from lysis with IGEPAL CA-630 alone or IGEPAL CA-630 in RT mix. Error bars represent standard deviation (SD). Mean and SD values are shown on top of the corresponding bars.
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f1: Comparison of five different lysis solutions for small number of MDA-MB-468 breast cancer cells.Three replicate samples of approximately one hundred cells were subjected to each different lysis solution, RT-qPCR was run and threshold cycle (Ct) values were determined for RPL32 and EGFR. The earliest Ct values of IGEPAL CA-630/BSA indicate that it performed the best. Conversely, the latest Ct values for samples lysed in water indicate that it performed the worse. Lysis with BSA alone led to earlier Ct values than those obtained from lysis with IGEPAL CA-630 alone or IGEPAL CA-630 in RT mix. Error bars represent standard deviation (SD). Mean and SD values are shown on top of the corresponding bars.

Mentions: Transcript levels of ribosomal protein L32 (RPL32) and epidermal growth factor receptor (EGFR), which are highly expressed in the MDA-MB-468 cell line, were measured by RT-qPCR. Highly expressed genes were chosen to avoid issues with stochastic gene expression, which can be a problem when working with very small numbers of cells. Sample preparation and the subsequent RT-qPCR were performed simultaneously to avoid batch-to-batch variation. The results shown in Fig. 1 are the average of the three replicates.


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

Le AV, Huang D, Blick T, Thompson EW, Dobrovic A - Sci Rep (2015)

Comparison of five different lysis solutions for small number of MDA-MB-468 breast cancer cells.Three replicate samples of approximately one hundred cells were subjected to each different lysis solution, RT-qPCR was run and threshold cycle (Ct) values were determined for RPL32 and EGFR. The earliest Ct values of IGEPAL CA-630/BSA indicate that it performed the best. Conversely, the latest Ct values for samples lysed in water indicate that it performed the worse. Lysis with BSA alone led to earlier Ct values than those obtained from lysis with IGEPAL CA-630 alone or IGEPAL CA-630 in RT mix. Error bars represent standard deviation (SD). Mean and SD values are shown on top of the corresponding bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525356&req=5

f1: Comparison of five different lysis solutions for small number of MDA-MB-468 breast cancer cells.Three replicate samples of approximately one hundred cells were subjected to each different lysis solution, RT-qPCR was run and threshold cycle (Ct) values were determined for RPL32 and EGFR. The earliest Ct values of IGEPAL CA-630/BSA indicate that it performed the best. Conversely, the latest Ct values for samples lysed in water indicate that it performed the worse. Lysis with BSA alone led to earlier Ct values than those obtained from lysis with IGEPAL CA-630 alone or IGEPAL CA-630 in RT mix. Error bars represent standard deviation (SD). Mean and SD values are shown on top of the corresponding bars.
Mentions: Transcript levels of ribosomal protein L32 (RPL32) and epidermal growth factor receptor (EGFR), which are highly expressed in the MDA-MB-468 cell line, were measured by RT-qPCR. Highly expressed genes were chosen to avoid issues with stochastic gene expression, which can be a problem when working with very small numbers of cells. Sample preparation and the subsequent RT-qPCR were performed simultaneously to avoid batch-to-batch variation. The results shown in Fig. 1 are the average of the three replicates.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia [2] Translational Genomics &Epigenomics, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia.

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.

No MeSH data available.


Related in: MedlinePlus