Mircury lna pcr primers

Due to limited sample quantity, BALB/c validation was not possible. Validation of miR-150–5p and miR-23a was performed in 24h samples for C57BL/6 mice at 1 Gy, 2 Gy, 4 Gy, 8 Gy and sham irradiated. No 48 h samples were available for C57BL/6 mice. Validation of miR-150–5p and miR-23a-3p was done in 24 h and 48 h samples for C3H/HeJ mice at 1 Gy, 2 Gy, 4 Gy, 8 Gy and sham irradiated. For miR-92a-5p, miR-99a-5p and miR-223a-5p were performed in 48 h samples for C3H/HeJ mice at 1 Gy, 2 Gy, 4 Gy, 8 Gy and sham irradiated. Since 48 h and 72 h showed the highest differential expression between sham and irradiated animals, we chose this time for the C3H/HeJ mice. Three samples from each time and dose were used for qRT-PCR. Samples were validated using the miRCURY LNA RT Kit (Qiagen, Cat. No./ID 339340) for cDNA synthesis and miRCURY LNA SYBR Green PCR Kit (Qiagen, Cat. No./ID 339345) per manufacturer protocols. The following miRCURY LNA PCR primers were purchased from Qiagen (Cat. No./ID 339306): miR-16–5p, miR-23a-3p, miR-92a-5p, miR-99a-5p, and miR-223a-5p. Reactions were conducted in ABI QuantStudio and analyzed with miR-16–5p as a known endogenous control (45 (link)). Fold change was produced using this formula:
$${\mathrm{d}\mathrm{C}\mathrm{T}}_{\text{miRNA\_sampleA}}={\mathrm{C}\mathrm{T}}_{\text{miRNA\_sampleA}}-{\mathrm{C}\mathrm{T}}_{\text{miR}-16-5\text{p\_sampleA}}$$
$${\mathrm{d}\mathrm{d}\mathrm{C}\mathrm{T}}_{\text{miRNA\_sampleA}}={\mathrm{d}\mathrm{C}\mathrm{T}}_{\text{miRNA\_sampleA}}-{\mathrm{d}\mathrm{C}\mathrm{T}}_{\text{miRNA\_control}\text{sample}}$$
$${\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{d}\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}}_{\text{miRNA\_sampleA}}=2^{-\text{ddCTmiRNA\_sampleA}}$$

Total RNA, isolated from bulk populations of 50,000 spatially-sorted hepatocytes (n = 3) per FACS gate, was diluted to 5 ng/μL, and cDNA was reverse-transcribed using the miRCURY LNA RT Kit (Qiagen, cat. no. 339340) according to the manufacturer’s instructions on an Applied BioSystems ProFlex PCR System. Plates were prepared using the miRCURY SYBR Green PCR Kit (Qiagen, cat. no. 339346) with custom miRCURY LNA PCR primers (Qiagen cat. no. 339306, Supplementary Table 10). Each 10 μL reaction volume contained 5 μL 2x miRCURY SYBR Green Master Mix, 0.5 μL ROX reference dye, 1 μL PCR primer mix, 0.5 μL RNAse-free water and 3 μL of cDNA sample diluted 1:60. qPCR reactions and measurements were performed on a StepOne Real-Time PCR System (Thermo Fisher, cat. no. 4376357) according to the manufacturer’s instructions. Correlations between qRT-PCR and microarray measurements were calculated on a mouse-by-mouse basis.