The miRCat® Small RNA Cloning Kit is based upon a pre-activated, adenylated RNA linkering method and allows researchers to clone miRNAs and other small RNAs from primary RNA sources. This method permits cloning from any RNA source of any species. The miRCat-33® kit is a conversion of the miRCat Kit for the purpose of carrying out 5′ ligation–independent small RNA cloning.

MicroRNAs

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in post-transcriptional gene regulations [1]. Experimental evidence is rapidly accumulating that shows miRNAs play key roles in processes such as cellular differentiation, cell death, and cell metabolism. An miRNA is composed of a highly conserved core sequence of 21–23 nucleotides (the mature miRNA) contained within a less well conserved precursor sequence (pre-miRNA) ranging in size from 60 nucleotides to more than 120 nucleotides. This pre-miRNA sequence is part of a larger primary transcript that may contain a single pre-miRNA or two or more pre-miRNAs arranged as paired or polycistronic transcripts. Following transcription, pre-miRNAs form a characteristic stem-loop structure that is processed by the RNase III enzyme DROSHA [2] in concert with accessory proteins such as PASHA and DGCR8 [3, 4]. The pre-miRNA is then exported from the nucleus and is further processed by the DICER/RISC complex which releases the mature miRNA to carry out its regulatory function.

A number of investigators have reported methods for cloning miRNAs from primary RNA sources [5-10]. The miRCat® Small RNA Cloning kit is based upon a pre-activated, adenylated RNA linkering method and allows researchers to clone miRNAs and other small RNAs from primary RNA sources.

RNA Isolation and Enrichment

RNA species in the 18 to 26 nucleotide size range are purified from total RNA. Best results are obtained if 50–100 µg of total RNA is used; however, cloning can be performed with less mass if RNA is scarce. This size range contains mature microRNA sequences. Several options are available for purification, including denaturing PAGE, the miRVana® kit (Ambion®), or the flashPAGE® fractionator (Ambion®).

Cloning Linker Attachment

The 3′ and 5′ cloning linkers are ligated to purified small RNA species in preparation for cDNA synthesis and amplification.

Amplification and Cloning

Reverse transcription of the linkered RNA species is carried out followed by PCR amplification and cloning. Two cloning options are available. The preferred option is a SAGE-like method where the small RNA cloning units (miRNA + linkers) are serially ligated (concatemerized) and then cloned. This method is more efficient when using sequencing platforms with long read lengths. The second option is to directly clone the PCR amplicons. In both options, cloning can be done using any available PCR cloning vectors.

Sufficient materials are provided in the kit to generate more than ten small RNA libraries. The two most important aspects of miRNA cloning are the quantity and quality of the starting RNA and the maintenance of relative mass relationships during the Cloning Linker Attachment Phase. Total cellular RNA can be used to clone small RNA species but the absolute mass of small RNAs is very small and larger RNA species will compete for linker molecules. For this reason, it is best to prepare a highly enriched and purified small RNA fraction at the outset.

Once purified small RNA species are obtained, it is crucial to use sufficient linker mass to ensure efficient 3′ and 5′ linker attachment. We strongly encourage using the 3′ and 5′ linkers in the amounts and the concentrations called for in the Cloning Linker Attachment Phase. Reductions in the mass of linker in either of the linker steps will result in a substantial reduction in linkering efficiencies.

References

1.Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2):281–297.

2.Lee RC, Feinbaum RL, and Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 75(5):843–854.

3.Gregory RI et al. (2004) The Microprocessor complex mediates the genesis of microRNAs. Nature 432(7014):235–240.

4.Denli AM et al. (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014):231–235.

5.Berezikov E, Cuppen E, and Plasterk RH (2006) Approaches to microRNA discovery. Nat Genet 38 Suppl:S2–S7.

6.Cummins JM et al. (2006) The colorectal microRNAome. Proc Natl Acad Sci U S A, 103(10):3687–3692.

7.Elbashir SM, Lendeckel W, and Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15(2):188–200.

8.Lau NC et al. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science, 294(5543):858–862.

9.Pfeffer S, Lagos-Quintana M, and Tuschl T (2003) Cloning of small RNA molecules. Current protocols in Molecular Biology. p 26.4.1–26.4.18.

10.Sunkar R and Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell, 16(8):2001–2019.

11.England TE, Gumport RI, and Uhlenbeck OC (1977) Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase. Proc Natl Acad Sci U S A, 74:4839–4842.

12.Unrau PJ and Bartel DP (1998) RNA-catalysed nucleotide synthesis. Nature, 395:260–263.