Ncode express sybr greener mirna qrt pcr kit

To validate Illumina deep sequencing data, we determined the profiles of miRNAs by quantitative PCR (qPCR) analysis using miRNA-specific LNA primers (Exiqon). In brief, the purified small RNA was converted to cDNA using NCode™VILO™miRNA cDNA Synthesis Kit (Invitrogen) to optimize reverse transcription efficiency from the small RNAs. The resulting cDNA was then used as a template for qPCR analyses using NCode™EXPRESS^®SYBR GreenER™miRNA qRT-PCR Kit (Invitrogen) on the ABI Prism 7900HT. We initially evaluated whether the most common reference RNAs used in qPCR experiments, such as 5S and 12S rRNAs, tRNA, and U6 snRNA, showed uniform level across the samples by plotting the raw C_T values for each samples. Our variance analysis in expression levels of each of the reference RNAs showed that U6 snRNA was the most consistently expressed RNA, followed by tRNA, 12S, and 5S rRNAs. Thus, the relative level of miRNAs was normalized to that of U6. The final results of qPCR in relative level were expressed as the ratio of miRNA in the injured nerve to that in the uninjured nerve using the 2^−ΔΔCT method. To verify miRNA-specific primer specificity, we confirmed a single peak in the melting-curve analysis, as well as run the reaction products on a 5% agarose gel (NuSieve 3: 1; Lonza) for all genes analyzed.

qRT‐PCR assay for mRNA analysis was performed using SYBR Green Mastermix (Affymetrix, Sunnyvale, CA, USA) on a Bio‐Rad iQ5 thermal cycler (Bio‐Rad Laboratories, Portland, ME, USA). The evaluation of relative expression levels was carried out by the comparative cycle threshold (CT) method using housekeeping gene GAPDH as a control. For miRNA expression analysis, total RNA including small RNAs was extracted using the miRNeasy Mini Kit (Qiagen), and cDNA was synthesized using an NCode miRNA First‐Strand cDNA Synthesis Kit (Life Technologies). qRT‐PCR was performed using an NCode Express SYBR GreenER miRNA qRT‐PCR Kit (Life Technologies) on a Bio‐Rad iQ5 thermal cycler. The relative expression level of U6 snRNA was used to normalize miRNA expression in each sample. The sequences of primers used for amplification are listed in Table 1.

One microgram of total RNA was converted to cDNA using the NCode VILO miRNA cDNA Synthesis Kit (Life Technologies)followed by qPCR using the NCode EXPRESS SYBR GreenER miRNA qRT-PCR kit (Life Technologies). The cycles passing threshold were recorded and the expression of miRNAs was calculated relative to U6B, a ubiquitously expressed small nuclear RNA. The forward primer for miR-124 was 5′- TAAGGCACGCGGTGAATGCC-3′. The forward primer for miR-924 was 5′-CCTCTGCCCTCTAAAGGTTTGC-3′. The forward primer for U6B was 5′-CGCAAGGATGACACGCAAATTCG-3′. The reverse primer was the NCode miRNA universal qPCR primer (Invitrogen). Data are presented as target miRNA or mRNA expression=2ΔCt, with ΔCt=(U6B or GAPDH Ct – target miRNA or mRNA Ct). qRT-PCR was carried out in triplicate for each sample for both the U6B or GAPDH control and each miRNA or mRNA.