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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 Gesú 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.

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Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).
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fig2: Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).

Mentions: The same samples were also checked with Agilent 2100 Bioanalyzer which is one of the most versatile microfluidics-based platforms for the analysis of DNA, RNA, proteins and cells. In all electropherograms the 28S and 18S RNAs are represented on the right side (Figure 2), and the smaller species (LMW RNAs) are present at a very low concentration and distinguishable on the left side of the profile (see the magnification of the LMW RNAs region in Figure 2). In the electropherogram, all samples present a similar HMW profile region, but the major differences are recognizable in the LMW region. While total RNA recovery is quite good, and similar for the three protocols (> 1.2 μg/μl), the LMW fractions are substantially different (Table 1). In particular, TRIzol reagent allowed the highest LMW RNA recovery (22%–34% of total RNA), while RNEasy Mini Kit the lowest (2.5%–3%). MirVana miRNA Isolation Kit gives good yields for LMW RNA species (16%–19%) even before the enrichment step. While LMW RNA species extracted with TRIzol and MirVana have comparable profiles (Figure 2); RNEasy kit retains only one RNA peak in comparable concentrations to the others (peak a in the magnification of Figure 2). Therefore, the lab-on-a-chip analysis is a useful tool to quantify precisely the amount of LMW RNAs of samples extracted with different protocols.


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)

Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).
Mentions: The same samples were also checked with Agilent 2100 Bioanalyzer which is one of the most versatile microfluidics-based platforms for the analysis of DNA, RNA, proteins and cells. In all electropherograms the 28S and 18S RNAs are represented on the right side (Figure 2), and the smaller species (LMW RNAs) are present at a very low concentration and distinguishable on the left side of the profile (see the magnification of the LMW RNAs region in Figure 2). In the electropherogram, all samples present a similar HMW profile region, but the major differences are recognizable in the LMW region. While total RNA recovery is quite good, and similar for the three protocols (> 1.2 μg/μl), the LMW fractions are substantially different (Table 1). In particular, TRIzol reagent allowed the highest LMW RNA recovery (22%–34% of total RNA), while RNEasy Mini Kit the lowest (2.5%–3%). MirVana miRNA Isolation Kit gives good yields for LMW RNA species (16%–19%) even before the enrichment step. While LMW RNA species extracted with TRIzol and MirVana have comparable profiles (Figure 2); RNEasy kit retains only one RNA peak in comparable concentrations to the others (peak a in the magnification of Figure 2). Therefore, the lab-on-a-chip analysis is a useful tool to quantify precisely the amount of LMW RNAs of samples extracted with different protocols.

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 Gesú 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