Mir 30d

Five μL of miRNAs (for sera) or 100 ngs (for tumor and normal mammary gland) were reverse transcribed into cDNAs in a final volume of 30 μL using a Taqman miRNA reverse transcription kit (Applied Biosystems, Grand Island, NY, USA). qPCR was performed using Taqman universal PCR mix (Applied Biosystems) and specific primers. Primers for U6 (#001973), miR-486 (#001278), miR-202 (#001195), and miR-30d (#000420) were purchased from Applied Biosystems. Each amplification reaction was performed in duplicate in a final volume of 20 μl with two μl of cDNA. qPCR reactions of sera from healthy subjects and patients with metastatic breast cancer for a particular probe were in the same plate to limit mechanical errors. The expression levels of miR-486 were normalized to miR-202 (mouse sera and mammary gland), U6 (cardiac and skeletal muscle), and miR30d (C2C12 cells) using 2- ^ΔΔCt method. In case of C2C12cells, one microgram of miRNA preparation was used for cDNA synthesis. mRNAs from C2C12 cells were prepared using RNAeasy kit from Qiagen and subjected to qRT-PCR using the primers that amplify both Ank1 and sAnk1 (primer sequences; forward: 5′ GAC GCA TGA CCT ACA GTC TTC 3′ and reverse: 5′ GCT ATC CTC TCC CTT CTT CTC T 3′) and βActin (forward: 5′ AAT GAG GCC GAG GAC TTT GAT TGC 3′ and reverse: 5′ AGG ATG GCA AGG GAC TTC CTG TAA 3′).