Wells A1 to G12 (1–84) each contain a miScript Primer Assay for a pathway/disease/functionally related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells H9 and H10 contain replicate reverse transcription controls (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC).
Wells A1 to P12 (1–372) each contain a miScript Primer Assay for a pathway/disease/ functionally related mature miRNA. Wells P13 and P14 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells P15 to P20 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells P21 and P22 contain replicate miRTC miScript Primer Assays (miRTC). Wells P23 and P24 contain replicate positive PCR controls (PPC).
Pathway-Focused miScript miRNA PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96-well formats A, C, D, and F.
Wells 1 to 84 each contain a miScript Primer Assay for a pathway/disease/functionally related gene. Wells 85 and 86 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells 87 to 92 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells 93 and 94 contain replicate miRTC miScript Primer Assays (miRTC). Wells 95 and 96 contain replicate positive PCR controls (PPC). Wells 97–100 are empty.
Wells 1 and 2 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Well 3 of each row contains a miR-16 miScript Primer Assay (16). Well 4 of each row contains a miR-21 miScript Primer Assay (21). Well 5 of each row contains a miR-191 miScript Primer Assay (191). Wells 6 to 8 of each row each contain an assay for a different snoRNA (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 9 and 10 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 8 cDNA samples.
Wells 1 and 2 contain replicate C. elegans miR-39 miScript Primer Assays. Well 3 contains a miR-16 miScript Primer Assay. Well 4 contains a miR-21 miScript Primer Assay. Well 5 contains a miR-191 miScript Primer Assay. Wells 6 to 8 each contain an assay for a different snoRNA (Well 6 = SNORD61 assay, Well 7 = SNORD95 assay, Well 8 = SNORD96A assay). Wells 9 and 10 contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 contain replicate positive PCR controls (PPC). This pattern is repeated 7 additional times from wells 13 to 96. Wells 97 to 100 are empty. This format enables the quality assessment of up to 8 cDNA samples.
Total RNA was isolated from HCT 116 colorectal cancer cells that had been treated with ± 5-aza-2'-deoxycytidine (5-aza-2'-dC) demethylation reagent. This reagent irreversibly inhibits DNA methyltransferase driven methylation reactions by incorporating into DNA and covalently binding to the active site of the DNA methyltransferase. cDNA was prepared with miScript HiSpec Buffer using the miScript II RT Kit. The Human miRNome miScript miRNA PCR Array, in combination with the miScript SYBR® Green PCR Kit, was used to profile mature miRNA expression by real-time PCR. Data analysis was performed using the online miScript miRNA PCR Array Data Analysis Tool. [A] A scatter plot of 2-ΔCT values demonstrates that significant differences exist in the mature miRNA expression levels of the 2 samples. [B] A volcano plot shows regulated miRNAs (±4-fold is used as a cutoff and indicated by red lines). 104 miRNAs were strongly upregulated and 30 were strongly downregulated in 5-aza-2'-dC treated HCT 116 cells. When a p value of 0.05 is applied (indicated by blue line), the upregulation of 89 of the 104 miRNAs is significant, and the downregulation of 21 of the 30 miRNAs is significant.
Scatter plots show the relative miRNA expression levels from pooled RNA from healthy and colon cancer serum samples, characterized using miScript miRNA PCR Arrays.
