Figure 1: In vitro RNA transcription process

Product advantages

High yield: can produce up to 200 μg in 2 hours through transcription reaction

Better versatility: suitable for transcription of 20nt-10000nt RNA

Excellent performance: effectively reduce the byproducts of transcription during IVT process

Multiple applications: can be used for ordinary, labeled, and modified RNA synthesis

Product Properties

Figure 2: Yield of transcripts of different lengths

Figure 3: Quality of transcripts of different lengths

 

Figure 4: Expression of transcribed mRNA in HEK-293 cells

 

Note:The mRNA has been capped with Vaccinia Capping Enzyme(Yeasen#10614/10615).

Experimental methods

Thawing reagents

Centrifuge the T7 RNA Polymerase Mix briefly and place it on ice. Thaw 10×Transcription Buffer and ribonucleotides (ATP, CTP, GTP, UTP), mix and centrifuge to the bottom of the tube, place 10×Transcription Buffer at room temperature, and place 4 types of ribonucleotides on ice.

B.Assembly transcription reaction at room temperature

Prepare the reaction system according to the following system:

Components	Volume (µL)	Final concentration
RNase free H2O	Up to 20	-
10×Transcription Buffer	2	1×
CTP / GTP/ ATP/ UTP (100 mM each)	2 each	10 mM each
Template DNA	1 µg	-
T7 RNA Polymerase Mix	2	-

 

Notes:

1. The reaction is configured at room temperature. Since 10× Transcription Buffer contains spermidine, the high concentration of spermidine maycause DNA template precipitation at low temperature.
   For Short transcripts (<100 nt), 2 µg template can be used, transcription time extended to 4-8 hs.
   3. For long transcripts (>1000 nt), recommended to use linearized plasmids as templates for transcription.
   4. Perform the reaction in the PCR instrument with the hot lid open to prevent the evaporation.
   5. The reaction product may have a white precipitate. The precipitate is the magnesium pyrophosphate produced by the free pyrophosphate and magnesium ions in the reaction solution which does not affect the subsequent experiments. If you want to remove it, you can add some EDTA. If the addition of EDTA affects subsequent experiments, the supernatant can also be recovered by centrifugation.
   6. Keep the reagents and containers without RNase contamination.

C.Incubate at 37°C for 2 hours  

Mix the components solution, briefly centrifuge to the bottom of the tube, and incubate at 37°C for 2 hs. If the transcript length is less than 100 nt, extend the reaction time to 4-8 hs.  

D.DNase I treatment (optional)  

After the reaction is completed, add 2 μL of DNase I (RNase free) to each tube and incubate at 37°C for 15 mins to remove the template DNA.

Frequently Asked Questions

1. Low transcript yield

The quality of template is closely related to the yield. The yield of experimental group is significantly lower than the control group. The possible reasons are:

• The experimental template contains inhibitory components;
• The template maybe has something wrong.

Suggestions: ① Re-purify the template; ② Determine the template quantification and its integrity; ③ Extend the reaction time; ④ Increase the amount of template input; ⑤ Use other promoters and other RNA polymerases.

2. Low yield of short transcripts

Short template fragments can inhibit the reaction. When the transcription product is less than 100 nt, if you want to increase the yield, extend the reaction time to 4-8 hs or increase the amount of template to 2 μg.

3. RNA transcription length is larger than expected

If electrophoresis result shows that the product band is larger than the expected size, possible reasons may be: ①The plasmid template may not be completely linearized; ②The 3' end of the sense strand has a prominent structure; ③The RNA has a secondary structure which is not completely denatured.

Suggestions: ①Check whether the template is completely linearized, and if necessary, perform additional linearization; ②Select a suitable restriction enzyme to avoid 3' overhangs, or use Klenow Fragment /T4 DNA polymerase to complete the transcription before proceeding; ③Use denatured gel to detect RNA products.

4. RNA transcription length is smaller than expected

If the electrophoresis shows that the product band is smaller than the expected size, the possible reasons are: ①The template contains a termination sequence similar to T7 RNA polymerase terminator; ②The GC content of the template is too high.

Suggestions: ①Lower the reaction temperature (for example, 30°C). Sometimes lowering the temperature can contribute to extend the transcription length, but it may reduce the yield. Try other RNA polymerases for transcription; ②If the template GC content is high, perform the reaction at 42℃, or add SSB to increase the yield and transcription length.

5. Electrophoretic tailing of transcriptional products

Tailing during electrophoresis may be caused by: ① RNase contamination during the experimental operation; ②The DNA template is contaminated with RNases.

Suggestions: ①Use RNase-free pipette tips and EP tubes, wear disposable latex gloves and masks, and all reagents are prepared with RNase free H₂O. ②Re-purify the template DNA.

Ordering information

Product name	Catalog number	Specification
T7 High Yield RNA Synthesis Kit	10623ES	50/100/500 T
T7 RNA Polymerase GMP-grade (250 U/μL)	10625ES	10KU/100KU/2500KU/25MU
Pyrophosphatase, Inorganic GMP-grade (1 U/μL)	10620ES	10U/100U/1000U
Murine RNase inhibitor GMP-grade (40 U/μL)	10621ES	10KU/20KU/100KU/1MU
mRNA Vaccinia Capping Enzyme GMP-grade	10614ES	2KU/10KU/100KU
mRNA Cap 2'-O-Methyltransferase GMP-grade	10612ES	10KU/50KU/250KU
Deoxyribonuclease I (DNase I) GMP-grade	10611ES	500/2000/10000 U
Hieff NGS® RNA Cleaner	12602ES	1/5/60/450 mL
Hieff NGS® mRNA Isolation Master Kit	12603ES	24/96 T