What’s in the kit:
List of Kit contents:
TGIRT Kit contents (10 reactions or 25 reactions):
TGIRT®-III enzyme, 1 x 10 ml (KTGIRT-10) or 3 x 10 ml (KTGIRT-25)
10X Primer mix, 50 ml (PM-110)
10 x DTT, 50 ml (D-121)
5 x Reaction Buffer, 100 ml (RX-120)
- The kit includes the TGIRT®-III enzyme, a mixture of RNA and DNA oligonucleotides that can be annealed to form the R2 RNA/R2R DNA heteroduplex containing the first adapter sequences, dithiothreitol (DTT), and reaction buffer to carry out TGIRT® template-switching reactions for Illumina RNA-seq and ssDNA-seq analysis.7,8,9,10,12 The kit enables synthesis of cDNA copies of RNA templates with the first RNA-seq adapter seamlessly linked to the 5’ end of the cDNA. For RNA-seq library construction, a DNA oligonucleotide (purchased separately) containing the second RNA-seq adapter sequences must be added to the 3' end of the cDNA with thermostable 5'AppDNA/RNA ligase (New England BioLabs; M0319S or M0319L) prior to PCR amplification.
What experiments can I do?
1. Comprehensive strand-specific transcriptome profiling.8
2. RNA-seq of whole-cell, exosomal, plasma, and other cell-free RNAs.7,8,15
3. Profiling of miRNA, tRNA and other small non-coding RNAs.1-9,12,15
4. RIP-seq, HITS-CLIP, irCLIP and CRAC for characterization of RNA-protein interactions, and ribosome profiling.1,10,115.
5. Identification of RNA base modifications by high-throughput sequencing.4,5,13,14
6. Single-stranded DNA-seq.16
7. Analysis of FFPE tumor samples (consult InGex technical support).
Here’s why you should use TGIRT:
- Higher thermostability, processivity, and fidelity than retroviral reverse transcriptases, allowing full-length, end-to-end cDNA synthesis from highly structured and/or heavily modified RNAs (e.g., tRNAs), and RNAs containing GC-rich repeat expansions.1-9,12,15,17
- Novel end-to-end template-switching activity that enables attachment of RNA-seq or PCR adapters during reverse transcription and eliminates the need for a separate tailing or RNA 3'-adapter ligation step.1 This template-switching activity greatly facilitates strand-specific RNA-seq library construction with less bias than procedures employing random hexamer primer or using RNA ligase for adapter ligation.1,7,8
- Ability to produce comprehensive profiles from RNA input as low as 2 ng.7,15
- RNA-seq library construction from RNA template to PCR products takes less than 5 hours.7,8,15
TGIRT is better for RNA-seq library construction via TGIRT-template switching:
1. Comprehensive strand-specific transcriptome profiling.
TGIRT®-seq of ribodepleted, fragmented Universal Human Reference RNA samples recapitulates the relative abundance of human transcripts and spike-ins comparably to non-strand-specific TruSeq v2 and better than strand-specific Tru-Seq v3. TGIRT®-seq is significantly more strand-specific than TruSeq v3 and eliminates sampling biases from random hexamer priming that are inherent to TruSeq. TGIRT®-seq shows more uniform 5’ to 3’ gene coverage and identifies more splice junctions than TruSeq. TGIRT®-seq enables simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq as structured small ncRNAs, including tRNAs, which are essentially absent from TruSeq datasets.8
2. RNA-seq of human whole-cell, exosomal, plasma, and other extracellular RNAs.
Fast processing time (< 5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); comprehensive transcript profiles including mRNAs and lncRNAs together with small ncRNAs, including full-length reads of tRNAs, pre-miRNAs, and other structured small ncRNAs; does NOT require RNA ligase; less biased and more efficient with greater strand specificity than conventional methods.7,8,15
3. RNA-seq of highly structured and/or heavily modified RNA templates.
Higher thermostability, processivity and strand-displacement makes it possible to obtain full-length, end-to-end cDNAs of tRNAs and other structured/modified small ncRNAs, which are refractory to retroviral RTs.4-9,12-15.
4. RNA-seq library construction via TGIRT® template-switching in methods like RIP-seq, HITS-CLIP, irCLIP, CRAC, ribosome profiling.
Fast processing time (< 5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); does NOT require RNA ligase, less biased and more efficient by having fewer steps in the procedure.1,10,11
5. ssDNA-seq of human plasma and E. coli genomic DNAs.
Captures precise DNA ends with a simpler workflow by initiating DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin.16
- Mohr, S., Ghanem, E., Smith, W., Sheeter, D., Qin, Y., King, O., Polioudakis, D., Iyer, V.R., Hunicke-Smith, S. Swamy, S., Kuersten, S., and Lambowitz, A.M. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958-970, 2013.
- Collins, K. and Nilsen, T. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology. RNA 19, 1017-1018, 2013.
- Enyeart, P.J., Mohr, G., Ellington, A.D., and Lambowitz A.M. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. Mobile DNA 5:2, 2014.
- Katibah, G.E., Qin, Y., Sidote, D.J., Yao, J., Lambowitz, A.M. and Collins, K. Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5. Proc. Natl. Acad. Sci. U.S.A. 111, 12025-12030, 2014.
- Shen, P.S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M.H., Cox, J., Chen, Y., Lambowitz, A.M., Weissman, J.S., Brandman, O., and Frost, A. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75-78, 2015.
- Zheng, G., Qin, Y., Clark, W.C., Yi, C., He, C., and Lambowitz, A.M. and Pan, T. Efficient and quantitative high-throughput transfer RNA sequencing. Nat. Methods 12, 835-837, 2015.
- Qin,Y., Yao,J., Wu,D., Nottingham, R., Mohr, S, Hunicke-Smith, S., Lambowitz, A.M., High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases. RNA 22, 111-128, 2016.
- Nottingham, R.M., Wu, D.C., Qin, Y., Yao, J., Hunicke-Smith, S., and Lambowitz, A.M. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22, 597-613, 2016.
- Burke, J.M., Kincaid, R.P., Nottingham, R.M., Lambowitz, A.M., and Sullivan, C.S. DUSP11 activity on tri-phosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady state levels of cellular non-coding RNAs. Genes & Dev. 30, 2071-2092, 2016.
- Zarnegar, B.J., Flynn, R.A., Shen, Y., Do, B.T., Chang, H.Y. and Khavari, P.A. irCLIP platform for characterization of protein-RNA interactions. Nat. Methods 13, 489-492, 2016.
- Haque, N. and Hogg, J.R. Easier, better, faster, stronger: Improved methods for RNA-protein interaction studies, Mol. Cell, 2016 http//dx.doi.org/10.1016/j.molcel.2016.05.019
- Bazzini, A.A., del Viso, F., Moreno-Mateos, M.A., Johnstone, T.G., Vejnar, C.E., Qin, Y., Yao, J., Khokha, M.K., and Giraldez, A.J. Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition. EMBO J. 35, 2087-2103, 2016.
- Clark, W.C., Evans, M.E., Dominissini, D., Zheng, G., and Pan, T. tRNA base methylation identification and quantification via high-throughput sequencing. RNA 22, 1771-1784, 2016.
- Liu et al. ALKBH-mediated tRNA demethylation regulates translation. Cell 167, 816-828, 2016,
- Shurtleff, M.J., Yao, J., Qin, Y., Nottingham, R.M., Temoche-Diaz, M., Schekman, R., and Lambowitz, A.M. A broad role for YBX1 in defining the small non-coding RNA composition of exosomes. Proc. Natl. Acad. Sci. U.S.A. 114, 8987-8995, 2017.
- . Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching. Scientific Reports 7, 8421, 2017.
- . Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase. Nucleic Acids Res. 46, e1, 2018.
IMPORTANT PATENT AND TRADEMARK INFORMATION:
1. The TGIRT® RTs and their Template-Switching Activity are the subject of U.S. patent 9,012,183 entitled “Use of template switching for DNA synthesis”, issued April 21, 2015 and United States Patent 1,0150,955 entitled “Stabilized Reverse Transcriptase Fusion Proteins" issued December 11, 2018 and additional U.S. and foreign patents and patent applications licensed exclusively to InGex, LLC by the University of Texas. The TGIRT®--III RT is the subject of U.S. patent #7,670,807 entitled “RNA-Dependent DNA Polymerase from Geobacillus stearothermophilus”, issued March 2, 2010 and licensed exclusively to InGex, LLC by East Tennessee State University. Use of TGIRT® RTs or the TGIRT® template-switching kit for commercial purposes is subject to the following limited license from InGex, LLC.
2. NOTICE TO PURCHASER: LIMITED LICENSE:
The purchase of this product includes a limited, non-transferable license to use this amount of the TGIRT® RT kit product and practice the TGIRT® template-switching technology, which is claimed in issued, pending, and planned U.S. and international patent applications exclusively licensed to InGex, LLC, and patents issuing therefrom, solely for the purchaser’s research activities. No use in diagnostic procedures is authorized. The license granted herein is personal to the original purchaser and may not be transferred to any other party outside the purchaser’s company or university. No other right or license is conveyed or granted either expressly, by implication or by estoppel, to resell or repackage this product. Certain other restrictions and limitations may apply as set forth in warranty statements and other documentation provided with the purchase of TGIRT® RT or the TGIRT® Template-Switching RNA-Seq kit. Further information can be obtained by contacting the InGex, LLC. For additional information, please contact InGex through the Tech Support form listed at the top of the page.
3. TGIRT® is Registered in the U.S. Patent and Trademark Office. Registration number: 4,835,933