Small RNA Cloning Procedure (FWD)

Introduction

The small RNA cloning procedure is based on adapter ligation. The adapter oligonucleotides are used for priming reverse transcription and for defining the orientation and sequence of the cloned small RNAs. This protocol is isotope free, utilises unmodified small RNAs and is routinely used to characterise miRNAs and siRNAs from various plant tissues. The total nucleic acid (TNA) isolation is adapted from White and Kaper (1989). Our small RNA cloning protocol results from modifications of protocols originally published by the Tuschl, Bartel and Carrington groups (Elbashir et al., 2001; Pfeffer et al., 2003; Lau et al., 2001; Llave et al., 2002) and may be cited as Chappell et al., 2005.

Small RNA cloning protocol

The gel purified small RNAs are ligated directly to a non-phosphorylated 5’-adapter oligonucleotide using T4 RNA ligase. The ligation products are separated from the excess of 5’-adapter on a 15% denaturing polyacrylamide gel and are subsequently ligated to a 5’-phosphorylated 3’-adapter oligonucleotide with a blocked 3’-hydroxyl terminus. The final ligation products are separated from the excess of 3’-adapter and are subjected to reverse transcription and PCR amplification. The gel purified PCR products are digested with EcoRI and NcoI restriction enzymes and subsequently concatamerised using T4 DNA ligase. The concatamers are ligated into an EcoRI-NcoI digested cloning vector and then TOP10 cells are transformed with the recombinant plasmids. Individual colonies are screened for the size of concatamer inserts by PCR and selected PCR fragments are purified and submitted for sequencing. The small RNA sequences are extracted from the sequence manually or automatically using software tools (e.g., Staden Package or software developed in-house).

From :David Baulcombe's website