Contributions
Abstract: PB1511
Type: Publication Only
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Quantitative polymerase chain reaction (qPCR) is a rapid and sensitive approach to identify miRNA expression. However, because of the shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA. Different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of qPCR of miRNA. In CML there is not a standard control gene, both U6 and SNORD95 have been evaluated with discordant results. So, its mandatory to evaluate another housekeeping gene to use in these pathology.
Aims
To evaluate Mir-30-b as housekeeping gene in miRNA qPCR in CML cell line and patient´s samples
Methods
mirVana miRNA Isolation Kit (Ambion®) was used for RNA extraction from peripheral blood mononuclear cells (PBMCs) from 5 patients at diagnosis and 4 healthy donors’ samples (DS) and K562 CML cell line previously incubated with either imatinib 1000nM and 450nM or nilotinib 120nM or 60nM according to calculated IC50 using MTT assay. For qPCR a protocol that is designed to specifically amplify and quantify miRNAs using stem-loop primers was used. SNORD95, U6 and Mir-30-b were compared.
Results
in human samples U6 and miR30-b had acceptable values to be used as control gene: standard derivation (SD) was 1,38 and 1,48 for U6 and 1,8 and 2,3 for mIR-30 in CML samples and DS respectively; while coefficient of variation (CV) was 5,8% for U6 and 6,1% for Mir-30 CML samples and 7,2% and 9,9% for DS respectively. Two-way anova comparison between CML and DS was not statistically significant neither was it comparing each ARN group individually. (p> 0,05)
In K562 cell line SD was 1,2 for SNORD95; 1,7 for U6; and 0,2 for Mir-30. CV was 52% for SNORD 95, 65% for U6, and 12% for mir-30. Tukey's multiple comparisons test of each group of RNA with different treatment showed statistical significance difference (p<0,001)
Conclusion
In conclusion, miR-30 is a suitable control gene when evaluating miRNAs expresión in K562 cell line and for human samples. To confirm this findings, a bigger cohort should be evaluated. U6 was a good control gene in human samples but not in CML cell line.
Keyword(s): Chronic myeloid leukemia, Molecular markers, Quantitative RT-PCR
Abstract: PB1511
Type: Publication Only
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Quantitative polymerase chain reaction (qPCR) is a rapid and sensitive approach to identify miRNA expression. However, because of the shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA. Different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of qPCR of miRNA. In CML there is not a standard control gene, both U6 and SNORD95 have been evaluated with discordant results. So, its mandatory to evaluate another housekeeping gene to use in these pathology.
Aims
To evaluate Mir-30-b as housekeeping gene in miRNA qPCR in CML cell line and patient´s samples
Methods
mirVana miRNA Isolation Kit (Ambion®) was used for RNA extraction from peripheral blood mononuclear cells (PBMCs) from 5 patients at diagnosis and 4 healthy donors’ samples (DS) and K562 CML cell line previously incubated with either imatinib 1000nM and 450nM or nilotinib 120nM or 60nM according to calculated IC50 using MTT assay. For qPCR a protocol that is designed to specifically amplify and quantify miRNAs using stem-loop primers was used. SNORD95, U6 and Mir-30-b were compared.
Results
in human samples U6 and miR30-b had acceptable values to be used as control gene: standard derivation (SD) was 1,38 and 1,48 for U6 and 1,8 and 2,3 for mIR-30 in CML samples and DS respectively; while coefficient of variation (CV) was 5,8% for U6 and 6,1% for Mir-30 CML samples and 7,2% and 9,9% for DS respectively. Two-way anova comparison between CML and DS was not statistically significant neither was it comparing each ARN group individually. (p> 0,05)
In K562 cell line SD was 1,2 for SNORD95; 1,7 for U6; and 0,2 for Mir-30. CV was 52% for SNORD 95, 65% for U6, and 12% for mir-30. Tukey's multiple comparisons test of each group of RNA with different treatment showed statistical significance difference (p<0,001)
Conclusion
In conclusion, miR-30 is a suitable control gene when evaluating miRNAs expresión in K562 cell line and for human samples. To confirm this findings, a bigger cohort should be evaluated. U6 was a good control gene in human samples but not in CML cell line.
Keyword(s): Chronic myeloid leukemia, Molecular markers, Quantitative RT-PCR