Limits...
Quantification of small non-coding RNAs allows an accurate comparison of miRNA expression profiles.

Masotti A, Caputo V, Da Sacco L, Pizzuti A, Dallapiccola B, Bottazzo GF - J. Biomed. Biotechnol. (2009)

Bottom Line: The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed.Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species.Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Microarrays Laboratory, Bambino GesĂș Children's Hospital, P. za S. Onofrio 4, 00165 Rome, Italy. andrea.masotti@opbg.net

ABSTRACT
MicroRNAs (miRNAs) are highly conserved approximately 22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3'-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

Show MeSH
Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2735750&req=5

fig1: Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).

Mentions: RNA samples extracted from HeLa, COS-1, and LCL were run on agarose gel to visualize the differences between various extraction methods. COS-1 RNA samples, extracted with TRIzol reagent and MirVana kit, clearly showed the High Molecular Weight (HMW) 28S and 18S rRNA bands, while LMW RNAs are visualized as faint, smeary bands (Figure 1, lanes 1 and 2, resp.). COS-1 extracted with RNeasy kit displayed only the HMW RNA bands (28S and 18S) (Figure 1, lane 3), while the enriched LMW fraction obtained with MirVana kit is clearly displayed in the lowest part of the gel (Figure 1, lane 4). Similar results (not shown) were also obtained with the other cell lines.


Quantification of small non-coding RNAs allows an accurate comparison of miRNA expression profiles.

Masotti A, Caputo V, Da Sacco L, Pizzuti A, Dallapiccola B, Bottazzo GF - J. Biomed. Biotechnol. (2009)

Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735750&req=5

fig1: Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).
Mentions: RNA samples extracted from HeLa, COS-1, and LCL were run on agarose gel to visualize the differences between various extraction methods. COS-1 RNA samples, extracted with TRIzol reagent and MirVana kit, clearly showed the High Molecular Weight (HMW) 28S and 18S rRNA bands, while LMW RNAs are visualized as faint, smeary bands (Figure 1, lanes 1 and 2, resp.). COS-1 extracted with RNeasy kit displayed only the HMW RNA bands (28S and 18S) (Figure 1, lane 3), while the enriched LMW fraction obtained with MirVana kit is clearly displayed in the lowest part of the gel (Figure 1, lane 4). Similar results (not shown) were also obtained with the other cell lines.

Bottom Line: The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed.Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species.Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Microarrays Laboratory, Bambino GesĂș Children's Hospital, P. za S. Onofrio 4, 00165 Rome, Italy. andrea.masotti@opbg.net

ABSTRACT
MicroRNAs (miRNAs) are highly conserved approximately 22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3'-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

Show MeSH