Cloning and characterization of Bubaline mammary miRNAs: An in silico approach.

Molecular Biology Reports
Krishnadeo Ankush KhadeBharat Bhushan

Abstract

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...Continue Reading

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Methods Mentioned

BETA
electrophoresis
PCR
restriction
Seq

Software Mentioned

BLAST
Ensembl

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