Synthesis of 2-thiouridine-modified (m)RNA
Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
RNT-109-S | 15 reactions x 20 μl | 295,00 | Add to Basket/Quote Add to Notepad |
RNT-109-L | 50 reactions x 20 μl | 595,00 | Add to Basket/Quote Add to Notepad |
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months after date of delivery
Description:
HighYield T7 mRNA Synthesis Kit (s2UTP) is designed to produce large amounts of thiouridine-modified (m)RNA via in vitro transcription with T7 RNA polymerase. The resulting internally modified (m)RNA can subsequently be used for microinjection, transfection or in vitro translation experiments.
Thiouridine modifications have been shown to increase (m)RNA stability and to reduce immunogenicity. A 5'-cap structure further increases translation efficiency. 5'-capping can be achieved by a) co-transcriptional capping with cap analoga (e.g. ARCA) or b) post-transcriptional capping with capping enzymes.
The kit contains sufficient reagents for 15 reactions (S-Pack) or 50 reactions (L-Pack) à 20 μl (7.5 mM GTP, 7.5 mM 2-Thio-UTP, 7.5 mM ATP, 7.5 mM CTP). An individual optimization of 2-Thio-UTP concentration can easily be achieved with the single nucleotide format.
A 20 μl reaction yields about 30-50 μg RNA after 30 min incubation (1 μg T7 control template, 1.4 kb RNA transcript). Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation)).
Content:
HighYield T7 RNA Polymerase Mix
RNT-109-S: 2x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)
RNT-109-L: 3x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)
HighYield T7 Reaction Buffer
1x 200 μl (10x), HEPES-based
ATP - Solution
1x 100 μl (100 mM)
GTP - Solution
1x 100 μl (100 mM)
CTP - Solution
1x 100 μl (100 mM)
UTP - Solution
1x 100 μl (100 mM)
2-Thio-UTP
RNT-109-S: 3x 10 μl (100 mM)
RNT-109-L: 8x 10 μl (100 mM)
T7 G-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class III phi6.5 promotor resulting in ~1400 nt RNA transcript
T7 A-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class II phi2.5 promotor (A-initiating) resulting in ~1400 nt RNA transcript
PCR-grade water
1x 1.2 ml
DTT
1x 100 μl (100 mM)
To be provided by user
T7 Promotor-containing DNA template
RNA purification tools
RNAse-free DNAse I
Important Notes (Read before starting)
Prevention of RNAse contamination
Although a potent RNase Inhibitor is included, creating a RNAse-free work environment and maintaining RNAse-free solutions is critical for performing successful in vitro transcription reactions. We therefore recommend
Template requirements
Minimum T7 promotor sequences:
T7 class III phi6.5 promotor (G-initiating)
5'-TAATACGACTCACTATAGNN…-3’
Bold: First base incorporated into RNA, NN: ideally CG
In vitro Transcription protocol
The general protocol is set up for 0.5 μg - 1 μg DNA template (refer to section 1.2 regarding template requirements), a final NTP concentration of 7.5 mM and 100% substitution of UTP by 2-Thio-UTP, respectively.
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. variation 2-Thio-UTP/UTP ratio , variation of template amount, variation of incubation time).
Component | Volume | Final conc. |
PCR-grade water | X μl | |
HighYield T7 Reaction Buffer (10x) | 2 μl | 1x |
DTT (100 mM) | 2 μl | 10 mM |
GTP (100 mM) | 1.5 μl | 7.5 mM |
2-Thio-UTP (100 mM) | 1.5 μl | 7.5 mM |
CTP (100 mM) | 1.5 μl | 7.5 mM |
ATP (100 mM) | 1.5 μl | 7.5 mM |
Template DNA | X μl | 1 μg |
HighYield T7 RNA Polymerase Mix | 2 μl | |
Total volume | 20 μl |
Please note: Reagents for the following steps are not provided within this kit.
DNA template removal
Depending on the down-stream application, removal of template DNA might be required. We recommend a salt-resistant, high efficiency DNAase such as Turbo™DNAse (ThermoFisher). Follow the manufacturer instructions.
Removal of 5'-triphosphate groups
5'-ends of in vitro phosphorylated RNAs carry a triphosphate group that is known to trigger RIG-1 mediated innate immune response in mammalian cells[1,2]. Removal with phosphatases (e.g. CIP) before final purification is therefore recommended for RNA probes intended for transfection experiments. Please refer to the following references for more detailed information: [1],[2].
(m)RNA purification
Purification of (m)RNA is required prior to transfection or (m)RNA quantitation by absorbance measurement. Spin column purification will remove proteins, salts and unincorporated nucleotides. Please follow the manufacturer instructions and ensure that the columns match with product size and possess a sufficient binding capacity (e.g. RNA Clean & Concentrator™ columns (Zymo Research) or Monarch® RNA Cleanup kit (NEB)). Other RNA purification methods such as LiCl precipitation may work but have not been tested.
(m)RNA quantitation
RNA concentration can be determined by absorbance measurement at 260 nm (A260) according to the Law-of-Lambert-Beer (A260 = 1 corresponds to 40 μg/ml ssRNA).
Related products: 2-Thio-UTP, #NU-1151
BIOZ Product Citations:
Selected References:
[1] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7) :e2005840.
[2] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3):367.