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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 between direct lysis and column-based RNA extraction on a cell dilution series.Direct lysis and column-based extraction were compared across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method, and assessed by RT-qPCR. Direct lysis gave at least 1 Ct earlier than the column-based method. Error bars represent SD. Mean and SD values are shown on top of the corresponding bars.
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f4: Comparison between direct lysis and column-based RNA extraction on a cell dilution series.Direct lysis and column-based extraction were compared across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method, and assessed by RT-qPCR. Direct lysis gave at least 1 Ct earlier than the column-based method. Error bars represent SD. Mean and SD values are shown on top of the corresponding bars.

Mentions: We then compared the two methods across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method. Raw Ct values obtained by RT-qPCR for RPL32 mRNA were used for the comparison. The relative effectiveness of the Arcturus PicoPure kit was consistent across the range of cell numbers tested, as reflected by the Ct values. At 10 and 100 cells in the dilution series, Ct values from directly lysed samples came up 1.8 cycles earlier than those from the Arcturus PicoPure column extraction method. At 1,000 cells, the difference in the mean Ct value was 1 Ct in favour of the lysis method (Fig. 4), indicating a possible decrease in efficiency of the lysis method at higher cell numbers. The difference in RNA yield, as measured by RT-qPCR, across the various cell numbers tested was statistically significant (pā€‰<ā€‰0.05).


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 between direct lysis and column-based RNA extraction on a cell dilution series.Direct lysis and column-based extraction were compared across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method, and assessed by RT-qPCR. Direct lysis gave at least 1 Ct earlier than the column-based method. Error bars represent 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

f4: Comparison between direct lysis and column-based RNA extraction on a cell dilution series.Direct lysis and column-based extraction were compared across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method, and assessed by RT-qPCR. Direct lysis gave at least 1 Ct earlier than the column-based method. Error bars represent SD. Mean and SD values are shown on top of the corresponding bars.
Mentions: We then compared the two methods across a range of cell numbers (10, 100, and 1,000 MDA-MB-468 cells), with two replicates at each cell number, for each method. Raw Ct values obtained by RT-qPCR for RPL32 mRNA were used for the comparison. The relative effectiveness of the Arcturus PicoPure kit was consistent across the range of cell numbers tested, as reflected by the Ct values. At 10 and 100 cells in the dilution series, Ct values from directly lysed samples came up 1.8 cycles earlier than those from the Arcturus PicoPure column extraction method. At 1,000 cells, the difference in the mean Ct value was 1 Ct in favour of the lysis method (Fig. 4), indicating a possible decrease in efficiency of the lysis method at higher cell numbers. The difference in RNA yield, as measured by RT-qPCR, across the various cell numbers tested was statistically significant (pā€‰<ā€‰0.05).

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