Shipping and Payments:
1. Do you ship overseas or to a P.O. Box?
InGex’s products are for research use only and therefore will only ship to recognized research institutions including universities and for-profit entities including established companies and start-up firms. InGex will NOT ship to a P.O. Box.
InGex ships to all locations worldwide using UPS or FedEx. If you are exempt from VAT or import duties please supply InGex with the appropriate forms at the time of your order, including but not limited to VAT exemption forms. Please note that InGex ships to international customers on MONDAYS ONLY.
2. What is your return/refund policy?
Orders may be cancelled by the customer prior to shipment. All sales of TGIRT products are final once the product ships and due to the nature of its products, InGex does not accept returns on its products. If a shipment is incorrect as a result of our error InGex will replace the product or refund the customer the cost of the product. If the incorrect order is a result of an error made by the purchasing agent, the product charges and shipping charges are the responsibility of the customer.
3. How should I store my products upon receipt?
You should store our products at -20 °C in order to minimize degradation of the product.
4. What form of payment do you accept?
We accept purchase orders from most companies and universities, and most major credit cards, and wired funds. InGex.com is a secure site and all data transmitted is encrypted to protect your credit card information. If you are a new U.S. account and wish to use a purchase order, we ask that you send us 3 credit references, a bank reference and a hard copy of your first purchase order so that we may set up your account. In general, we ask that first-time buyers outside of the U.S. use a credit card or wire transfer to prepay for their products.
For international payments via wire transfer, there will be an additional $25 added to the invoice to defray the cost of the Intermediary Bank Fee. The Intermediary Bank Fee is incurred upon transfer of your money into the United States. The intermediary bank in New York charges a $25 fee to transfer your money to our bank in St. Louis. Unfortunately, the $25 fee is taken from the money you wire us, which leaves you with an open balance on your invoice. Payments via credit card or check from a checking institution are not subject to these additional charges.
TGIRT Technical Questions:
TGIRT enzyme and kits
1. What are the difference between the TGIRT Improved Modular Template-Switching RNA-seq Kit and the original kit?
In the original kit, RT-110 is the mix of enzymes pre-bound with annealed R2 RNA/R2R DNA primers.
In this new kit, the previous RT-110 is split into individual components: the enzyme (KTGIRT-10 or 25) and mix of R2 RNA and R2R DNA primers (PM-110). Users will need to perform primer annealing before template-switching. This change is made to improve the efficiency of the reaction.
2. How many template-switching reactions does each TGIRT®-III kit contain?
Each kit contains materials for 25 reactions.
3. Should I purchase the TGIRT®-III kit or the stand-alone TGIRT®-III enzyme?
The TGIRT®-III kit contains the TGIRT®-III enzyme, mix of R2 RNA and R2R DNA primers, and reagents for users to conveniently carry out routine RNA-seq library construction using pre-designed Illumina adapter sequences.
The TGIRT®-III enzyme offers flexibility by allowing users to use custom adapter sequences. It is recommended that all oligonucleotides are ordered with PAGE purification.
1. Should I use Small RNA-seq/CircLigase or Total RNA-seq protocol?
The Small RNA-seq/CircLigase protocol is used for sequencing small RNAs, such as miRNAs. This procedure includes a gel purification step to select homogenously sized cDNA products after the template-switching reaction before circularizing with CircLigase.
The Total RNA-seq protocol is used for sequencing RNAs of different size classes, from small to long RNA templates, or a heterogenous mixture of both. This procedure does NOT require gel purification and therefore is faster and more efficient when working with small amounts of RNA templates. An optional size enrichment step by Agencourt AMPure XP beads (Beckman Coulter) or gel purification can be incorporated into the procedure after the template-switching reaction.
2. How much RNA templates should I use in template-switching reaction?
To optimally represent all RNA species in the sample pool, the R2 RNA/R2R DNA heteroduplex needs to be present in excess to the RNA templates. Therefore, the final concentration of RNA templates used in the template-switching reaction should not exceed 100 nM.
3. Can I use RNA templates with modified 5’ and 3’ ends in template-switching reaction?
TGIRT®-III enzyme requires RNA 3’ OH ends for optimal template-switches. It is recommended that users use T4 polynucleotide kinase to remove 3′ phosphates and 2′, 3′ cyclic monophosphates. Modifications at the 5’ ends of RNA templates do not affect the template-switching reaction.
Adapter Design and PCR
1. What is the Read 1 primer site in your Multiplex PCR primer and can I change it?
The Multiplex PCR primer in the current protocols contains the small RNA sequencing primer site used in the NEBNEXT Small RNA Library Prep Set for Illumina. It can be replaced by other primer sites. The sequence of corresponding R1R DNA oligonucleotide, which is used in the adenylation and ligation reactions, will need to be replaced at the same time. PCR primers and the R1R DNA are NOT included in the TGIRT®-III kit.
2. Can I use customized R2 RNA and R2R DNA oligonucleotides if I purchase the stand-alone TGIRT®-III enzyme?
The R2 RNA and R2R DNA can be customized to contain additional sequences or be replaced by other primer sites. The R2R DNA needs to be complementary to the R2 RNA, with at least one nucleotide overhang to base pair with the last nucleotide at the 3’ end of an RNA template. The overhang can be an equimolar mixture of A, T, G and C nucleotides to eliminate end biases or a single nucleotide complementary to the 3’ end of the target RNA to enrich for target RNA templates during the template-switching reaction. It is also desirable to keep the length of the R2R DNA at approximately 40 nt so that the unextended R2R DNA can be optimally cleaned up by the MinElute Reaction Cleanup Kit (Qiagen, Cat. No. 28204).
3. How many amplification cycles should I do during PCR?
To avoid accumulation of large amounts of adapter dimers, which can inhibit amplification of cDNA products and result in PCR artifacts, it is recommended to amplify for no more than 15 cycles.
4. How to clean up adapter dimers after PCR?
Agencourt AMPure XP beads (Beckman Coulter) can be used to clean up adapter dimers and to enrich for desired DNA sizes in the sample. The beads to sample volume ratio will need to be experimentally determined for enrichment of different DNA sizes.
Use TGIRTs for other applications
1. Can I use the TGIRT®-III enzyme for cDNA synthesis from a DNA primer annealed to an RNA template?
Yes. It is important to note that the Tm of the annealed DNA primer should be above 60°C, which is the operational temperature of the TGIRT®-III enzyme. For cDNA synthesis from polyA+ RNAs, this is accomplished by using an anchored (dT)42 primer. It is recommended that the RNA template with annealed DNA primer be pre-incubated with the TGIRT®-III enzyme in reaction buffer containing freshly added dithiothreitol (DTT) (see below) for 30 min at room temperature. The cDNA synthesis reaction is initiated by adding dNTPs and then raising the temperature to 60°C. The reaction buffer, particularly the NaCl concentration, and incubation time at 60°C should be optimized for the length, structure, and modification level of RNA templates (Zheng et al. 2015).
2. Can I use the TGIRT®-III enzyme for single-stranded DNA sequencing?
Yes. The protocol for ssDNA-seq via template-switching is very similar to RNA-seq, with a few changes to the adapters and the components of the reaction buffer, as described in Wu and Lambowitz, 2017.
The R2 RNA and R2R DNA primers in the TGIRT®-III kit for initiating the template-switching reaction are the same for both RNA-seq and ssDNA-seq. The R1R DNA, which always needs to be ordered separately, is different for ssDNA-seq. For all oligonucleotides ordered separately, it is recommended that they are ordered with PAGE purification.
3. What is the reaction buffer for the TGIRT®-III enzyme if I want to use it for other applications?
The standard 1X reaction buffer contains 450 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 5 mM MgCl2. A final concentration of 5 mM dithiothreitol (DTT) should be added separately to the reaction. The DTT should be prepared fresh or from a frozen stock solution. The 450 mM NaCl used in the template-switching reaction suppresses multiple template-switches to very low levels (Qin et al. 2016; Nottingham et al. 2016). The concentration of NaCl can be adjusted for other applications. For new applications, it is recommended that users test a range of NaCl concentrations from 25 to 450 mM NaCl.
1. After template-switching reaction, I can’t detect cDNA products on the gel.
To detect cDNA products on the gel, it is recommended to label the 5’ end of R2R DNA with [γ-32P]-ATP before the template-switching reaction. Alternatively, when using the unlabeled R2R DNA, portions or all of the cDNA products after the template-switching reaction can be cleaned up by MinElute Reaction Cleanup Kit (Qiagen, Cat. No. 28204), 5’ labeled with [γ-32P]ATP, and analyzed by denaturing PAGE.
2. After PCR amplification, I only see peaks corresponding to PCR primers on the Bioanalyzer trace.
The absence of adapter dimers, which are approximately 120-125 bp on the Bioanalyzer trace, indicates that both the unextended R2R DNA and the cDNA products synthesized during the template-switching reaction, are not ligated to the adenylated R1R DNA during the ligation reaction. The problem typically occurs when adenylated R1R DNA falls below expected concentration after cleanup. It is recommended to measure the concentration of adenylated R1R DNA before proceeding to the ligation reaction. To ensure consistent recovery of R1R DNA during cleanup by the Oligo Clean & Concentrator Kit (Zymo Research, Cat. No. D4060), multiple adenylation reactions can be combined prior to cleanup to increase elution volume.
3. After PCR amplification, I only see adapter dimers that are approximately 120-130 bp on the Bioanalyzer trace.
For optimal activity of the TGIRT®-III enzyme, dithiothreitol (DTT) needs to be freshly made or thawed from frozen aliquots. It is recommended to aliquot the DTT stock upon arrival of the TGIRT®-III kit.
A positive control, which can be a commercial RNA ladder or other RNA standards, can be included when working with small amounts of RNA templates. If desired peaks are detected for the control on the Bioanalyzer trace, clean up the adapter dimers and re-run the DNA library on the Bioanalyzer, as sometimes large amounts of adapter dimers can interfere with visualization of peaks corresponding to RNA templates. If there are still no visible peaks detected after cleaning up the adapter dimers, it is possible that the concentration of DNA library is below the detection limit of Bioanalyzer. The DNA library can be concentrated and re-analyzed by Bioanalyzer. Alternatively, the DNA library can be quantitated by qPCR to determine obtainable sequencing depth.
Wu, Douglas C. and Alan M. Lambowitz. 2017. "Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching." Scientific Reports 7 (1): 8421.
Nottingham, Ryan M., Douglas C. Wu, Yidan Qin, Jun Yao, Scott Hunicke-Smith, and Alan M. Lambowitz. 2016. “RNA-Seq of Human Reference RNA Samples Using a Thermostable Group II Intron Reverse Transcriptase.” RNA (New York, N.Y.) 22 (4): 597–613.
Qin, Yidan, Jun Yao, Douglas C. Wu, Ryan M. Nottingham, Sabine Mohr, Scott Hunicke-Smith, and Alan M. Lambowitz. 2016. “High-Throughput Sequencing of Human Plasma RNA by Using Thermostable Group II Intron Reverse Transcriptases.” RNA (New York, N.Y.) 22 (1): 111–28.
Zheng, Guanqun, Yidan Qin, Wesley C. Clark, Qing Dai, Chengqi Yi, Chuan He, Alan M. Lambowitz, and Tao Pan. 2015. “Efficient and Quantitative High-Throughput tRNA Sequencing.” Nature Methods 12 (9): 835–37.
updated: March 23, 2018