miRNA Cloning Products

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.

Documentation and Support

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.


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miRCat® small RNA cloning is based upon the pre-activated, adenylated RNA linking method that has been used successfully in many labs since its development in 2001 [1]. This method permits cloning from any RNA source in any species. The miRCat-33® Conversion Kit allows the miRCat Kit to be used for 5′ ligation-independent small RNA cloning using the method of Pak and Fire [2]. 

DescriptionPart #Pricing
miRCat® RNA Cloning Kit11-02-06-02$895.00 USDOrder

Material sufficient for ten cloning experiments is provided in the miRCat™ kit.

Kit Contents:

  • 3′ Linker 1 pre-activated, adenylated cloning linker
  • 5′ M.R.S. cloning linker
  • miSPIKE 21-mer internal RNA control
  • Forward and Reverse/RT primers
  • T4 RNA Ligase
  • Ligation buffer w/o ATP
  • Ligation Enhancer
  • 10mM ATP
  • T4 DNA Ligase
  • 3M NaOAc (pH 5.2)
  • 10mg/ml Glycogen
  • IDTE (pH 7.5)
  • IDT Water
  • Technical Manual
  • Edge Biosystems Gel Purification Columns
Figure 1

Figure 1. The Process of Cloning miRNAs and Other Small RNAs from any Total RNA Source Using the miRCat® Kit. The Ban I restriction enzyme sites in the cloning linkers are shown by cross hatching. The page numbers indicate the pages in the full length manual where each step of the protocol begins.


References

  1. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2):281–97.
  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):843–54.

miRCat® Small RNA Cloning Kit

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Each oligo pack contains:

  • 3′ pre-activated, adenylated cloning linker
    Sequence- 5′- rAppTGGAATTCTCGGGTGCCAAGG/ddC/ -3′
  • Replacement PCR Primer
    Sequence- 5′- CCTTGGCACCCGAGAATT -3′
DescriptionPart #Pricing
1 nm miRCat®-33 Conversion Oligo Pack51-01-13-09$175.00 USDOrder
5 nm miRCat®-33 Conversion Oligos Pack51-01-13-10$595.00 USDOrder

miRCat-33® is a conversion of the miRCat® kit for the purpose of carrying out 5′ ligation-independent small RNA cloning using the method of Pak and Fire [1]. Primer is included at no charge.


References

  1. 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):843–54.

miRCat-33® Conversion Kit

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454 Adaptor Primers are designed to convert the small RNA libraries generated by miRCat ligations (Set I) or by miRCat-33 ligations (Set II) into 454-compatible PCR libraries for deep sequencing [1].


References

  1. Denli AM, et al. (2004) Processing of primary microRNAs by the Microprocessor complex. Nature, 432(7014):231–5.

454 miRCat® and miRCat-33® Adapter Primers

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Linker-1 is the original modban sequence employed by Lau and Bartel in 2001 [1] and contains a Ban-I restriction site. Linker-2 contains Ava-I and Sty-I restriction sites. Linker-3 contains EcoR-I and Msp-I restriction sites and was adapted from Pfeffer and Tuschl [2]. All three linkers are modified with a 3′-terminal dideoxy-C (ddC) base to prevent self ligation. Experiments have shown miRNA linker performance can vary depending on the RNA source. For this reason, the miRNA Cloning Linker Pack – 3 Linker Types, 1 nmole each, provides small samples of each of the three linkers and is useful for optimizing methods before using precious RNA samples in large-scale library construction.Linker oligonucleotides are provided lyophilized and are ready for use in cloning; just resuspend at the desired concentration and add to your ligation mix (using T4 RNA Ligase without ATP). Use of this reagent can improve cloning efficiency of miRNAs, which have a 5′-phosphate and will circularize if attachment of linkers is attempted using RNA Ligase in the presence of ATP.

Modifications

  • 5′-adenylated; fully activated and ready for use
  • 3′-end blocked with a dideoxy-C base

Purification

  • HPLC

Quality Control

  • Identity confirmed by ESI mass spectrometry
  • Tested for reactivity with RNA ligase
  • Certificate of Analysis for each linker available online here
DescriptionSequencePart #Pricing
5 nm miRNA Cloning Linker 1/5rApp/CTGTAGGCACCATCAAT/3ddC/11-04-03-05$595.00 USDOrder
1 nm miRNA Cloning Linker 1/5rApp/CTGTAGGCACCATCAAT/3ddC/11-04-02-08$175.00 USDOrder
5 nm miRNA Cloning Linker 2/5rApp/CACTCGGGCACCAAGGA/3ddC/11-04-03-06$595.00 USDOrder
1 nm miRNA Cloning Linker 2/5rApp/CACTCGGGCACCAAGGA/3ddC/11-04-02-09$175.00 USDOrder
1 nm miRNA Cloning Linker 3/5rApp/TTTAACCGCGAATTCCAG/3ddC/11-04-02-10$175.00 USDOrder
5 nm miRNA Cloning Linker 3/5rApp/TTTAACCGCGAATTCCAG/3ddC/11-04-03-07$595.00 USDOrder
miRNA Cloning Linker 3-Pack – 3 Linkers, 1 nm each11-02-06-01$450.00 USDOrder

References

  1. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2):281–97.
  2. Gregory RI et al. (2004) The Microprocessor complex mediates the genesis of microRNAs. Nature, 432(7014):235–40.


3′ Cloning Linkers

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T4 RNA Ligase uses ATP to adenylate the 5′-end of a single-strand nucleic acid sequence. This activated adenylated-oligo is then covalently connected (ligated) to the 3′-OH of a second single-stranded sequence. Adenylated oligonucleotides containing a pyrophosphate linkage are substrates for T4 RNA Ligase in the absence of ATP [1]. IDT will custom adenylate an oligonucleotide for use with RNA-Ligase using the chemical adenylation method of Unrau and Bartel [2]. T4 RNA Ligase will use an adenylated DNA linker with similar efficiency as an adenylated RNA linker and IDT recommends use of adenylated DNA oligos for this application. Note that IDT requires blocking the 3′-end of an adenylated oligo so it cannot circularize; use of either 3′-Spacer C3 /3SpC3/ or dideoxycytosine /3ddC/ is preferred.

To order adenylated linkers, go to the Custom DNA Oligos page and order custom oligonucleotides with 5' adenylation as a modification.


References

  1. 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.
  2. Unrau PJ and Bartel DP (1998) RNA-catalysed nucleotide synthesis. Nature, 395:260–263.

Custom Adenylation

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The 5′ M.R.S Linker sequence is designed for use with any of the 3′ miRNA Cloning linkers. The sequence has been optimized for linking to the 5′ end of RNAs containing a 5′ phosphate group. The reaction is carried out with T4 RNA Ligase in the presence of 1 mM ATP. The sequence contains restriction endonuclease recognition sites compatible with Ban 1 (Linker 1), Sty I and Ava I (Linker 2) and EcoRI (Linker 3). Upon reverse transcription of doubly linked RNAs, the restriction endonuclease appropriate for the 3′ cloning linker will also generate compatible ends in the 5′ M.R.S Linker sequence permitting concatamerization and/or cloning. Further, the additional restriction sites in the M.R.S Linker, when matched with specific 3′ linkers can generate ends for directional cloning. For example, M.R.S Linker/Linker 3 digestion with Eco RI and Ban I will leave a BanI 5′ end and an EcoRI 3′ end.

DescriptionSequencePart #Pricing
1 nm M.R.S. miRNA Cloning LinkerTGGAATrUrCrUrCrGrGrGrCrArCrCrArArGrGrU11-04-02-12$175.00 USDOrder
5 nm M.R.S. miRNA Cloning LinkerTGGAATrUrCrUrCrGrGrGrCrArCrCrArArGrGrU11-04-03-08$595.00 USDOrder

5′ M.R.S (Multiple Restriction Site) miRNA Cloning Linker

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miSPIKE is a synthetic 21-mer RNA whose sequence does not match any known small RNA sequence in GenBank, miRBase, or RNAdb. The RNA oligonucleotide was synthesized without a 5′ phosphate so that the marker can serve as both a size marker for small RNA enrichment on a denaturing acrylamide gel and as a 3’ ligation control but is inert to 5’ ligation.

Sequence: 5′-rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU -3′

DescriptionPart #Pricing
100 pm miSPIKE™ Internal RNA Control51-01-03-10$25.00 USDOrder

miSPIKE 21-mer Internal RNA Control

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piSPIKE is a synthetic 31-mer RNA whose sequence is a ten nucleotide extension of miSPIKE and is also synthesized without a 5′ phosphate.

Sequence: 5′- rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrUrCrArCrUrGrArArCrGrU -3′

DescriptionPart #Pricing
100 pm piSPIKE™ Internal RNA Control51-01-16-44$35.00 USDOrder

piSPIKE 31-mer Internal RNA Control