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MicroRNA levels quantified in whole blood varies from PBMCs.

Atarod S, Smith H, Dickinson A, Wang XN - F1000Res (2014)

Bottom Line: To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs).The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood.Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

View Article: PubMed Central - PubMed

Affiliation: Haematological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

ABSTRACT
MicroRNAs are non-coding RNAs that negatively regulate mRNA expression and play significant roles in both health and disease. Differential microRNA expression has been used to aid diagnosis and discriminate disease stages. The accuracy and reliability of microRNA expression measurement is of utmost importance. Quantification of microRNA expression in human peripheral blood is commonly detected using total RNA extracted via different methods. To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs). This study examined miR-146a-5p and miR-155-5p expression using total RNA extracted in parallel from whole blood and PBMCs of 14 healthy volunteers. The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood. Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

No MeSH data available.


Normalised miRNA expression comparison from whole blood and PBMCs.(A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is significantly varied across all the three different groups (p=0.002). MicroRNA expression is higher in PBMCs extracted via either MP or MM method in comparison to whole blood. **p<0.01 and ns: not significant.
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f7: Normalised miRNA expression comparison from whole blood and PBMCs.(A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is significantly varied across all the three different groups (p=0.002). MicroRNA expression is higher in PBMCs extracted via either MP or MM method in comparison to whole blood. **p<0.01 and ns: not significant.

Mentions: With inclusion of stringent quality controls, we assessed miR-146a-5p and miR-155-5p expression in total RNA extracted in parallel from whole blood using PAXM (n=14) and PBMCs using MP (n=14) (Dataset c). Our results showed that there was no correlation (Figure 5, miR-146a-5p: r=-0.352, p=0.217 and miR-155-5p: r=0.380, p=0.180) between PAXM and MP in the expression of both miR-146a-5p and miR-155-5p. In a PCR reaction, it is assumed that the target expression doubles at every reaction cycle. Bland-Altman analysis also showed that the two methods did not agree as the bias was greater than 1 which equated to more than one qPCR cycle difference between the two methods (Figure 5A and 5B). Mookherjeeet al. had used MM to extract total RNA from PBMCs, then correlated miRNA expression between the PAXM and MM method (Mookherjee & El-Gabalawy, 2013). To eliminate the possibility that using MP was the reason for the non-correlation and disagreement, we tested the three different extraction methods (PAXM, MP and MM) for both miRNAs in a randomly selected cohort of five healthy volunteers (Figure 6) (Dataset d). The results demonstrated that PAXM and MP as well as PAXM and MM did not correlate nor agreed with one another. However, MP and MM methods agreed with each other and could therefore be interchanged as the bias between the two methods for both miR-146a-5p and miR-155-5p was only 0.769 (SD=0.307) and 0.892 (SD=0.802), respectively. Interestingly, normalised miRNA expression was significantly different only between PAXM and MM methods (miR-146a-5p and miR-155-5p: p<0.01). There was higher miRNA expression in PBMCs than in whole blood for both miRNAs (Figure 7).


MicroRNA levels quantified in whole blood varies from PBMCs.

Atarod S, Smith H, Dickinson A, Wang XN - F1000Res (2014)

Normalised miRNA expression comparison from whole blood and PBMCs.(A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is significantly varied across all the three different groups (p=0.002). MicroRNA expression is higher in PBMCs extracted via either MP or MM method in comparison to whole blood. **p<0.01 and ns: not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582824&req=5

f7: Normalised miRNA expression comparison from whole blood and PBMCs.(A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is significantly varied across all the three different groups (p=0.002). MicroRNA expression is higher in PBMCs extracted via either MP or MM method in comparison to whole blood. **p<0.01 and ns: not significant.
Mentions: With inclusion of stringent quality controls, we assessed miR-146a-5p and miR-155-5p expression in total RNA extracted in parallel from whole blood using PAXM (n=14) and PBMCs using MP (n=14) (Dataset c). Our results showed that there was no correlation (Figure 5, miR-146a-5p: r=-0.352, p=0.217 and miR-155-5p: r=0.380, p=0.180) between PAXM and MP in the expression of both miR-146a-5p and miR-155-5p. In a PCR reaction, it is assumed that the target expression doubles at every reaction cycle. Bland-Altman analysis also showed that the two methods did not agree as the bias was greater than 1 which equated to more than one qPCR cycle difference between the two methods (Figure 5A and 5B). Mookherjeeet al. had used MM to extract total RNA from PBMCs, then correlated miRNA expression between the PAXM and MM method (Mookherjee & El-Gabalawy, 2013). To eliminate the possibility that using MP was the reason for the non-correlation and disagreement, we tested the three different extraction methods (PAXM, MP and MM) for both miRNAs in a randomly selected cohort of five healthy volunteers (Figure 6) (Dataset d). The results demonstrated that PAXM and MP as well as PAXM and MM did not correlate nor agreed with one another. However, MP and MM methods agreed with each other and could therefore be interchanged as the bias between the two methods for both miR-146a-5p and miR-155-5p was only 0.769 (SD=0.307) and 0.892 (SD=0.802), respectively. Interestingly, normalised miRNA expression was significantly different only between PAXM and MM methods (miR-146a-5p and miR-155-5p: p<0.01). There was higher miRNA expression in PBMCs than in whole blood for both miRNAs (Figure 7).

Bottom Line: To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs).The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood.Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

View Article: PubMed Central - PubMed

Affiliation: Haematological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

ABSTRACT
MicroRNAs are non-coding RNAs that negatively regulate mRNA expression and play significant roles in both health and disease. Differential microRNA expression has been used to aid diagnosis and discriminate disease stages. The accuracy and reliability of microRNA expression measurement is of utmost importance. Quantification of microRNA expression in human peripheral blood is commonly detected using total RNA extracted via different methods. To date, no convincing data are available showing whether microRNA quantification results can be influenced by the use of total RNA extracted from whole blood or peripheral blood mononuclear cells (PBMCs). This study examined miR-146a-5p and miR-155-5p expression using total RNA extracted in parallel from whole blood and PBMCs of 14 healthy volunteers. The data showed that the quantification of miRNA using total RNA extracted from whole blood varied from that of PBMCs, indicating that the miRNA expression was a result of all the different cell-types present in whole blood. Our results suggested that the source of total RNA and the statistical analyses performed are crucial considerations when designing miRNA research.

No MeSH data available.