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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.


Related in: MedlinePlus

Changes in heat shock mRNA levels (hsp-16.2 and hsp-70, normalized to act-1) in C. elegans after heat treatment. The animals were raised at 20 °C, upshifted to 30 °C for 1 h and then returned to 20 °C. Error bar: standard deviation; n ≥ 5.
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fig0015: Changes in heat shock mRNA levels (hsp-16.2 and hsp-70, normalized to act-1) in C. elegans after heat treatment. The animals were raised at 20 °C, upshifted to 30 °C for 1 h and then returned to 20 °C. Error bar: standard deviation; n ≥ 5.

Mentions: To exemplify the usefulness of this method, we measured the expression levels of two heat-shock protein transcripts (hsp-16.2 and hsp-70) from single animals after induction via exposure to high temperature (Fig. 3).


Rapid RNA analysis of individual Caenorhabditis elegans.

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

Changes in heat shock mRNA levels (hsp-16.2 and hsp-70, normalized to act-1) in C. elegans after heat treatment. The animals were raised at 20 °C, upshifted to 30 °C for 1 h and then returned to 20 °C. Error bar: standard deviation; n ≥ 5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0015: Changes in heat shock mRNA levels (hsp-16.2 and hsp-70, normalized to act-1) in C. elegans after heat treatment. The animals were raised at 20 °C, upshifted to 30 °C for 1 h and then returned to 20 °C. Error bar: standard deviation; n ≥ 5.
Mentions: To exemplify the usefulness of this method, we measured the expression levels of two heat-shock protein transcripts (hsp-16.2 and hsp-70) from single animals after induction via exposure to high temperature (Fig. 3).

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.


Related in: MedlinePlus