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Rapid RNA analysis of individual Caenorhabditis elegans.

Ly K, Reid SJ, Snell RG - MethodsX (2015)

Bottom Line: RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze-thaw cycles may be needed to disrupt the cuticle before the cell contents are released.In addition, a large number of animals are required for successful RNA isolation.To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: •Faster and safer compared to conventional RNA extraction methods•Released RNA can be used directly for cDNA synthesis without purification•As little as a single worm is sufficient.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand.

ABSTRACT
Traditional RNA extraction methods rely on the use of hazardous chemicals such as phenol, chloroform, guanidinium thiocyanate to disrupt cells and inactivate RNAse simultaneously. RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze-thaw cycles may be needed to disrupt the cuticle before the cell contents are released. In addition, a large number of animals are required for successful RNA isolation. To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: •Faster and safer compared to conventional RNA extraction methods•Released RNA can be used directly for cDNA synthesis without purification•As little as a single worm is sufficient.

No MeSH data available.


Standard curve for act-1.Efficiency is 100.167%, calculated based on the formula: E = −1 + 10(−1/slope). The slope is −3.3179. The R2 is 0.9999.
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fig0005: Standard curve for act-1.Efficiency is 100.167%, calculated based on the formula: E = −1 + 10(−1/slope). The slope is −3.3179. The R2 is 0.9999.

Mentions: We demonstrate, using act-1 as a reference gene and absolute threshold cycle (Ct) values as an indirect measurement, that RNA is released from animals of all developmental stages from eggs to adults (Table 1). The Ct values decreased as expected from eggs to adults, corresponding to an increase in RNA amount. We found that as little as a single egg could produce Ct values within a useful working range. In addition, cDNA produced from each animal treatment (2 μL) is normally diluted to 25 μL and only 2.5 μL is used for each qPCR reaction, thus a single animal is sufficient for 10 qPCR. For a single adult worm, this resulted in a Ct value of 18.9 ± 0.28 for the act-1 transcript. The worm lysate did not inhibit cDNA synthesis and qPCR as the amplification efficiency for act-1 was 100.2% (Fig. 1). As expected increasing the number of animals in a lysate from 1 to 4, correspondingly decreased the Ct value. Therefore, it is possible to adjust several parameters (dilution factor, number of animals) to obtain Ct values suitable for different genes. In contrast, attempts to purify RNA from a single adult worm using Trizol were unsuccessful; no Ct value could be computed. When 100 adults were used in the Trizol method, sufficient RNA could be isolated as expected.


Rapid RNA analysis of individual Caenorhabditis elegans.

Ly K, Reid SJ, Snell RG - MethodsX (2015)

Standard curve for act-1.Efficiency is 100.167%, calculated based on the formula: E = −1 + 10(−1/slope). The slope is −3.3179. The R2 is 0.9999.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487333&req=5

fig0005: Standard curve for act-1.Efficiency is 100.167%, calculated based on the formula: E = −1 + 10(−1/slope). The slope is −3.3179. The R2 is 0.9999.
Mentions: We demonstrate, using act-1 as a reference gene and absolute threshold cycle (Ct) values as an indirect measurement, that RNA is released from animals of all developmental stages from eggs to adults (Table 1). The Ct values decreased as expected from eggs to adults, corresponding to an increase in RNA amount. We found that as little as a single egg could produce Ct values within a useful working range. In addition, cDNA produced from each animal treatment (2 μL) is normally diluted to 25 μL and only 2.5 μL is used for each qPCR reaction, thus a single animal is sufficient for 10 qPCR. For a single adult worm, this resulted in a Ct value of 18.9 ± 0.28 for the act-1 transcript. The worm lysate did not inhibit cDNA synthesis and qPCR as the amplification efficiency for act-1 was 100.2% (Fig. 1). As expected increasing the number of animals in a lysate from 1 to 4, correspondingly decreased the Ct value. Therefore, it is possible to adjust several parameters (dilution factor, number of animals) to obtain Ct values suitable for different genes. In contrast, attempts to purify RNA from a single adult worm using Trizol were unsuccessful; no Ct value could be computed. When 100 adults were used in the Trizol method, sufficient RNA could be isolated as expected.

Bottom Line: RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze-thaw cycles may be needed to disrupt the cuticle before the cell contents are released.In addition, a large number of animals are required for successful RNA isolation.To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: •Faster and safer compared to conventional RNA extraction methods•Released RNA can be used directly for cDNA synthesis without purification•As little as a single worm is sufficient.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand.

ABSTRACT
Traditional RNA extraction methods rely on the use of hazardous chemicals such as phenol, chloroform, guanidinium thiocyanate to disrupt cells and inactivate RNAse simultaneously. RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze-thaw cycles may be needed to disrupt the cuticle before the cell contents are released. In addition, a large number of animals are required for successful RNA isolation. To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: •Faster and safer compared to conventional RNA extraction methods•Released RNA can be used directly for cDNA synthesis without purification•As little as a single worm is sufficient.

No MeSH data available.