Cloning and characterization of Bubaline mammary miRNAs: An in silico approach
2019
Khade, Krishnadeo Ankush | Panigrahi, Manjit | Ahmad, Sheikh Firdous | Chauhan, Anuj | Kumar, Pushpendra | Bhushan, Bharat
MicroRNAs (miRNAs) are small non-coding RNAs ~ 19–25 nucleotides long that are involved in the regulation of gene expression. They negatively regulate the gene expression via inhibition or complete degradation of mRNAs by binding the complementary target sequences in 3′ untranslated region. The present investigation was aimed at profiling of miRNAs expressed in the Bubaline mammary tissue at dry stage of lactation cycle. Small RNAs were isolated from freshly collected mammary tissues and T4 RNA ligase was used to ligate the enriched miRNAs with 3′ and 5′ linker sequences in two separate reactions. cDNA copies were synthesized from linkered small RNAs followed by the PCR amplification. The PCR products were resolved on 15% non-denaturing polyacrylamide gel electrophoresis by gelstar staining. The PCR products were cloned using pGEM®-T easy vector system and the desired clones (with linkered small RNA sequences) were confirmed using restriction digestion of plasmids with EcoRI. Out of 15 Bubaline small RNA sequences, eight sequences (Seq. ID I–VIII) matched the size range of miRNA molecules i.e., 18–26 nucleotides. The Bubaline small RNA sequences II and III showed partial alignment with various mammalian and non-mammalian miRNAs. The small RNA sequences obtained in the present study did not show any perfect match with already reported mRNA, rRNA or tRNA sequences in different databases. Hence, only the Bubaline small RNA sequences that showed partial homology with miRNAs were considered as putative Bubaline miRNAs. The present study established the basic repertoire of miRNAs expressed at dry stage of lactation in Bubaline mammary gland.
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